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Pseudomonas aeruginosa is a prevalent gram-negative pathogenic bacterium ubiquitous in natural environment. Aquatic environment of wastewater serves as reservoirs of this bacteria and their wide resistance phenomenon to a number of antibiotics is frequently increasing. This study was conducted to determine the prevalence of P. aeruginosa in 10 industrial waste water and 10 tannery waste water samples of whole Dhaka and 65% (13/20) water samples were found positive for P. aeruginosa which was confirmed by both biochemical test & BiologTM Microbial Identification System. Kirby Bauer disc diffusion method was applied for antimicrobial susceptibility testing and isolates showed resistance to most of the commercial antibiotics except neomycin, gentamycin, streptomycin, ciprofloxacin and nalidixic acid, hence confirmed the multidrug resistance (MDR) of P. aeruginosa in wastewater which is one of the life-threatening public health issues all over the world causing ineffectiveness of several antibiotics. So, it is recommended to make sure surface water or food samples not to be contaminated by this antibiotic resistant P. aeruginosa that might be transferred to animal and human. In these circumstances, not only the hygiene practice is the first and foremost prerequisite but also management practices with effective wastewater disposal system can also be a part of awareness. Additionally, appropriate and logical use of antibiotics must be applied to reduce the emergence of multidrug pathogens to environment.

Pseudomonas aeruginosa is a prevalent gram-negative pathogenic bacterium ubiquitous in natural environment. Aquatic environment of wastewater serves as reservoirs of this bacteria and their wide resistance phenomenon to a number of antibiotics is frequently increasing. This study was conducted to determine the prevalence of P. aeruginosa in 10 industrial waste water and 10 tannery waste water samples of whole Dhaka and 65% (13/20) water samples were found positive for P. aeruginosa which was confirmed by both biochemical test & BiologTM Microbial Identification System. Kirby Bauer disc diffusion method was applied for antimicrobial susceptibility testing and isolates showed resistance to most of the commercial antibiotics except neomycin, gentamycin, streptomycin, ciprofloxacin and nalidixic acid, hence confirmed the multidrug resistance (MDR) of P. aeruginosa in wastewater which is one of the life-threatening public health issues all over the world causing ineffectiveness of several antibiotics. So, it is recommended to make sure surface water or food samples not to be contaminated by this antibiotic resistant P. aeruginosa that might be transferred to animal and human. In these circumstances, not only the hygiene practice is the first and foremost prerequisite but also management practices with effective wastewater disposal system can also be a part of awareness. Additionally, appropriate and logical use of antibiotics must be applied to reduce the emergence of multidrug pathogens to environment. [ABSTRACT FROM AUTHOR]

ABSTRACT Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii, combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.IMPORTANCEThe emergence of carbapenem-resistant Acinetobacter strains as a serious global health threat underscores the urgent need for effective treatment options. Although few drugs show promise against CR Acinetobacter infections, resistance to both drugs has been reported. In this study, the molecular characterization of spontaneous cefiderocol-resistant variants, a CR A. baumannii strain with antagonism to sulbactam, and an A. lwoffii strain with antagonism to avibactam, provides valuable insights into the mechanisms of resistance to cefiderocol. Some mechanisms observed are associated with mutations affecting efflux pumps, regulators, and iron homeostasis genes. These findings highlight the importance of understanding resistance mechanisms to optimize treatment options. They also emphasize the importance of early evaluation of drug synergies to address the challenges of antimicrobial resistance in Acinetobacter infections.

The rapid progress in point-of-care testing (POCT) has become a promising decentralized patient-centered approach for the control of infectious diseases, especially in resource-limited settings. POCT devices should be inexpensive, rapid, simple operation and preferably require no power supply. Here, we developed a simple bacterial sensing platform that can be operated by a smartphone for bacteria identification and antimicrobial susceptibility testing (AST) based on using a polydiacetylene (PDA) arrayed membrane chip. Each PDA array produced a unique color ‘fingerprint’ pattern for each bacteria based on different modes of action of toxins from bacteria on biomimetic lipid bilayers within PDA-lipid assemblies. We show that the PDA-based device can detect viable cells of bacteria as low as 104 ​CFU/mL within 1.5 ​h compared with several days of conventional bacterial identification, with the aid of a smartphone app. The device can also be used for an antimicrobial susceptibility test (AST) for at least two broad-spectrum antimicrobials within 4 ​h and provide identification of antimicrobial susceptibility and resistance, enabling the selection of appropriate therapies. This PDA-based sensing platform provides an alternative way for bacterial detection and could be used as a portable and inexpensive POCT device for the rapid detection of bacterial infection in limited-resource settings.

Chronic wounds, defined as those which remain open and inflamed for greater than six weeks, are a major area of clinical concern. Resulting in thousands of amputations per year and billions of dollars spent globally in treatment, chronic wounds are notoriously difficult to successfully treat. Two hallmarks of chronic wounds are that they are thought to harbor biofilm-associated bacteria and tend to be polymicrobial. While the research literature has repeatedly demonstrated the effects of biofilms on wound persistence and the changes to the efficacy of antibiotics, few studies have demonstrated what effect the polymicrobial condition has on the antibiotic tolerance of bacteria. To further explore this, four species of clinically relevant wound pathogens (Pseudomonas aeruginosa, Acinetobacter baumanii, Staphylococcus aureus, and Enterococcus faecalis) were tested in mono- and polymicrobial conditions using the current gold-standard methods for determining antibiotic susceptibility. Noticeable differences in antibiotic tolerance were observed in the polymicrobial condition, including both increased and decreased susceptibility, depending on the antibiotic used. Our data demonstrate that the current clinical methods used for testing antibiotic susceptibility can generate results that are not representative of the infection environment, which may contribute to treatment failure and persistence of polymicrobial infections. [ABSTRACT FROM AUTHOR]

External Quality Assessment schemes (EQAS) are mandatory to ensure quality standards in diagnostic methods and achieve laboratory accreditation. As host institution for two German culture-based bacteriology EQAS (RV-A and RV-B), we investigated the obtained data of 590 up to 720 surveys per year in RV-A and 2,151 up to 2,929 in RV-B from 2006 to 2023. As educational instruments, they function to review applied methodology and are valuable to check for systemic- or method-dependent failures in microbiology diagnostics or guidelines. Especially, containment of multi-resistant bacteria in times of rising antibiotic resistance is one major point to assure public health. The correct identification and reporting of these strains is therefore of high importance to achieve this goal. Moreover, correct antimicrobial susceptibility testing (AST) per se is important for selecting appropriate therapy, to restrict broad-spectrum antibiotics and minimize resistance development. The reports of participating laboratories displayed a high level of correct identification results in both schemes with mostly consistent failure rates around 2.2% (RV-A) and 3.9% (RV-B) on average. In contrast, results in AST revealed increasing failure rates upon modification of AST requirements concerning adherence to standards and subsequent bacterial species-specific evaluation. Stratification on these periods revealed in RV-A a moderate increase from 1.3% to 4.5%, while in RV-B failure rates reached 14% coming from 4.3% on average. Although not mandatory, subsequent AST evaluation and consistent reporting are areas of improvement to benefit public health.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx). [ABSTRACT FROM AUTHOR]

Aim: The study aimed to evaluate the bacterial and fungal profiles in Otitis Media (OM), Acute Otitis Media (AOM), and Chronic Otitis Media (COM) and the sensitivity patterns to antibiotics available in our hospital settings. Materials and Methods: A total of 150 clinically diagnosed cases of OM (AOM or COM) with ear discharge were enrolled. Swabs were cultured for microbial flora. Drug susceptibility testing was conducted using the Kirby-Bauer disc diffusion method. Results: The most common bacteria isolated in AOM was Streptococcus spp., and in COM it was Staphylococcus aureus. Among fungal isolates, Candida albicans dominate. The antimicrobial profile of the organisms revealed maximum sensitivity to Fluoroquinolones. Conclusions: Correct diagnosis and precise antibiotic prescription reduce the load of antibiotic resistance. [ABSTRACT FROM AUTHOR]

The occurrence of antimicrobial resistance due to the use of antimicrobials is considered to be a main cause for treatment failure of bacterial infections in humans and animals. The right of German veterinarians to use and prescribe medications such as antimicrobials is regulated by the Regulation of Veterinary Pharmacies (TÄHAV). The aim of this study was to investigate the impact of the second amendment to the TÄHAV in 2018 on the occurrence of antimicrobial resistance in selected bacterial pathogens isolated from dogs and cats in Germany. For this purpose, we analyzed antimicrobial susceptibility data from 38 German small animal practices gathered between 2015 and 2021 in cooperation with Laboklin (Labor für klinische Diagnostik GmbH & Co.KG, Bad Kissingen, Germany). Annual cumulative susceptibility data of eight bacterial species were analyzed and compared. The mean value of resistant isolates was determined for each year and supplemented by 95% confidence intervals. Encouraged by the amendment, an increase in sample submissions was observed in Germany. The highest resistance rates to the analyzed substances penicillin G, ampicillin, amoxicillin-clavulanic acid, cefovecin, and enrofloxacin were found for Staphylococcus pseudintermedius (S. pseudintermedius), S. aureus, and Escherichia coli (E. coli). In contrast, resistance rates were low for Pasteurella multocida (P. multocida) and β-hemolytic streptococci. Significant resistance trends (p < 0.05) assumed as influenced by the TÄHAV amendment could be the significant decreases in resistance rates of S. pseudintermedius against penicillin G to 67% (n = 322/479), and ampicillin to 63% (n = 286/453), as well as S. felis against amoxicillin-clavulanic acid and cefovecin to 2% (n = 2/109), furthermore, the reduction in the occurrence of resistance of S. aureus against enrofloxacin to 4% (n = 3/76) in 2021. Moreover, for all species, the efficacy against the analyzed substances was maintained over the study period. [ABSTRACT FROM AUTHOR]

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development. [ABSTRACT FROM AUTHOR]

The abusive use of antimicrobial compounds and the associated appearance of antimicrobial resistant strains are a major threat to human health. An improved antimicrobial administration involves a faster diagnosis and detection of resistances. Antimicrobial susceptibility testing (AST) are the reference techniques for this purpose, relying mainly in the use of culture techniques. The long time required for analysis and the lack of reproducibility of these techniques have fostered the development of high-throughput AST methods, including electrochemical biosensors. In this review, recent electrochemical methods used in AST have been revised, with particular attention on those used for the evaluation of new drug candidates. The role of nanomaterials in these biosensing platforms has also been questioned, inferring that it is of minor importance compared to other applications. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values (p value < 0.001) compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations. [ABSTRACT FROM AUTHOR]

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3–4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Drug-resistant bacterial strains seriously threaten human health. Rapid screening of antibiotics is urgently required to improve clinical treatment. Conventional methods of antimicrobial susceptibility testing rely on turbidimetry that is evident only after several days of incubation. The lengthy time of the assay can delay clinical treatment. Here, we proposed a single-cell level rapid system based on a microfluidic chip. The detection period of 30 min to 2 h was significantly shorter than the conventional turbidity-based method. To promote detection efficiency, 16 independent channels were designed, permitting the simultaneous screening of 16 drugs in the microfluidic chip. Prepositioning of drugs in the chip permitted prolonged transportation and storage. This may allow for the widespread use of the novel system, particularly in the regions where medical facilities are scarce. The growth curves were reported rapidly through a custom code in Matlab after tracking and photographing the bacteria during microscopy examination. The capability of the proposed system was validated by antimicrobial susceptibility testing trials with standard strains. The system provides a potentially useful detection tool for drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Recently, considerable uncertainty has arisen concerning the appropriate susceptibility testing for cefiderocol in gram-negative bacilli, particularly in the context of its application to Acinetobacter spp. The optimal method for assessing the susceptibility levels of Acinetobacter spp. to cefiderocol remains a subject of debate due to substantial disparities observed in the values obtained through various testing procedures. This study employed four minimum inhibitory concentration (MIC) methodologies and the disk diffusion to assess the susceptibility of twenty-seven carbapenem resistant (CR)- Acinetobacter strains to cefiderocol. The results from our study reveal significant variations in the minimum inhibitory concentration (MIC) values obtained with the different methods and in the level of agreement in interpretation categories between the different MIC methods and the disk diffusion test. Among the MIC methods, there was relatively more consistency in reporting the interpretation categories. For European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, the categorical agreement (CA) for MIC methods ranged between 66.7 and 81.5%. On the other hand, the essential agreement (EA) values were as low as 18.5–29.6%. The CA between MIC methods and disk diffusion was 81.5%. These results emphasize the need for a reliable, accurate, and clinically validated methodology to effectively assess the susceptibility of Acinetobacter spp. to cefiderocol. The wide variability observed in our study highlights the importance of standardizing the susceptibility testing process for cefiderocol to ensure consistent and reliable results for clinical decision-making. • Cefiderocol MIC values vary significantly across methods. • Differences in the agreement on interpreting categories were identified. • More consistency in reporting interpretation categories among MIC methods was seen. • The variability observed calls for standardization of cefiderocol testing. [ABSTRACT FROM AUTHOR]

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) comprises a global threat to humans and animals. Here, we report and characterize the MRSA t304/ST6 variant which, to our knowledge, represents the first case found in bovine clinical mastitis. In general, the MRSA t304/ST6 variant is rarely described in livestock, contrary to humans where it is widely recognized. Phenotypic and genotypic resistance profiling showed that the bovine-MRSA t304/ST6 isolate expressed low susceptibility toward cefoxitin (MICcefoxitin = 16 µg/mL) and carried the mecA resistance gene in the SCCmec IVa. The bovine-MRSA t304/ST6 isolate carried a plasmid similar to that which has been frequently observed among human-MRSA t304/ST6 isolates in Denmark (GenBank accession no. NZ_CP047022). In addition, a Staphylococcus prophage 3 (ϕSA3) was detected, encoding an immune evasion cluster (IEC) of putative virulence genes associated with human host-specificity (sea, sak, and scn). Taken together, these findings suggest that the MRSA t304/ST6 found in this study represents a recent host-jump event, with human to cow transmission. This study emphasizes the importance of and the need for performance of antimicrobial resistance surveillance among bovine mastitis pathogens, including S. aureus and MRSA. [ABSTRACT FROM AUTHOR]

Cefiderocol, a siderophore-cephalosporine conjugate antibiotic, shows promise as a therapeutic option for carbapenem-resistant (CR) Acinetobacter infections. While resistance has already been reported in A. baumannii , combination therapies with avibactam or sulbactam reduce MICs of cefiderocol, extending its efficacy. However, careful consideration is necessary when using these combinations. In our experiments, exposure of A. baumannii and A. lwoffii to cefiderocol and sulbactam or avibactam led to the selection of cefiderocol-resistant strains. Three of those were subjected to whole genome sequencing and transcriptomic analysis. The strains all possessed synonymous and non-synonymous substitutions and short deletions. The most significant mutations affected efflux pumps, transcriptional regulators, and iron homeostasis genes. Transcriptomics showed significant alterations in expression levels of outer membrane proteins, iron homeostasis, and β-lactamases, suggesting adaptive responses to selective pressure. This study underscores the importance of carefully assessing drug synergies, as they may inadvertently foster the selection of resistant variants and complicate the management of CR Acinetobacter infections.IMPORTANCEThe emergence of carbapenem-resistant Acinetobacter strains as a serious global health threat underscores the urgent need for effective treatment options. Although few drugs show promise against CR Acinetobacter infections, resistance to both drugs has been reported. In this study, the molecular characterization of spontaneous cefiderocol-resistant variants, a CR A. baumannii strain with antagonism to sulbactam, and an A. lwoffii strain with antagonism to avibactam, provides valuable insights into the mechanisms of resistance to cefiderocol. Some mechanisms observed are associated with mutations affecting efflux pumps, regulators, and iron homeostasis genes. These findings highlight the importance of understanding resistance mechanisms to optimize treatment options. They also emphasize the importance of early evaluation of drug synergies to address the challenges of antimicrobial resistance in Acinetobacter infections., Competing Interests: The authors declare no conflict of interest.

The occurrence of antimicrobial resistance due to the use of antimicrobials is considered to be a main cause for treatment failure of bacterial infections in humans and animals. The right of German veterinarians to use and prescribe medications such as antimicrobials is regulated by the Regulation of Veterinary Pharmacies (TÄHAV). The aim of this study was to investigate the impact of the second amendment to the TÄHAV in 2018 on the occurrence of antimicrobial resistance in selected bacterial pathogens isolated from dogs and cats in Germany. For this purpose, we analyzed antimicrobial susceptibility data from 38 German small animal practices gathered between 2015 and 2021 in cooperation with Laboklin (Labor für klinische Diagnostik GmbH & Co.KG, Bad Kissingen, Germany). Annual cumulative susceptibility data of eight bacterial species were analyzed and compared. The mean value of resistant isolates was determined for each year and supplemented by 95% confidence intervals. Encouraged by the amendment, an increase in sample submissions was observed in Germany. The highest resistance rates to the analyzed substances penicillin G, ampicillin, amoxicillin-clavulanic acid, cefovecin, and enrofloxacin were found for Staphylococcus pseudintermedius (S. pseudintermedius), S. aureus, and Escherichia coli (E. coli). In contrast, resistance rates were low for Pasteurella multocida (P. multocida) and β-hemolytic streptococci. Significant resistance trends (p < 0.05) assumed as influenced by the TÄHAV amendment could be the significant decreases in resistance rates of S. pseudintermedius against penicillin G to 67% (n = 322/479), and ampicillin to 63% (n = 286/453), as well as S. felis against amoxicillin-clavulanic acid and cefovecin to 2% (n = 2/109), furthermore, the reduction in the occurrence of resistance of S. aureus against enrofloxacin to 4% (n = 3/76) in 2021. Moreover, for all species, the efficacy against the analyzed substances was maintained over the study period.

Conducting antimicrobial susceptibility testing (AST) in a comparable manner requires the availability of a standardized method. Organizations, such as the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), provide standardized protocols for a range of fastidious bacteria but not for Mycoplasma hyorhinis. We developed a broth microdilution method for testing M. hyorhinis in a standardized and harmonized way using a modified Friis broth devoid of antimicrobial or otherwise bacterial growth-inhibiting agents. The type strain M. hyorhinis DSM 25591 was chosen to establish the methodology. The antimicrobial agents of interest were doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin, tested by using commercial SensititreTM microtiter plates. In addition, the suitability of the methodology was evaluated via variation of the individual ingredients of the modified Friis broth by either using different batches or choosing other distributors. Despite these alterations, the method provided reliable results. We obtained repeatable minimal inhibitory concentrations for all six tested field isolates and the M. hyorhinis type strain. With this newly proposed method, we aim to provide an improved AST method for diagnostic laboratories and monitoring purposes with better comparability between times and countries. In addition, this new method will allow for an improvement of targeted treatments using antimicrobial agents and thereby reduce the options for resistance development.

The aim of this study was to compare the reference broth microdilution (BMD) method with the Disk Diffusion (DD) test and strip test against a collection of 75 well-characterized Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) clinical strains to assess cefiderocol (CFD) antimicrobial activity. Whole-genome sequencing was performed on KPC-Kp strains by Illumina iSeq100 platform. The Categorical Agreement (CA) between the BMD method and DD test was 92% (69/75) with a Major Error (ME) of 16.7% (6/36). Additionally, the CA between the BMD method and test strip was 90.7% (68/75) with a Very Major Error (VME) of 17.9% (7/39) and 82.7% (62/75) between the strip test and DD with a ME of 30.2%. KPC-Kp strains showing resistance to CFD were 27 out of 75 (36%) by three methods. Specifically, 51.9% (14/27) of KPC-Kp resistant to CFD harbored blaKPC-3, while 48.1% (13/27) harbored mutated blaKPC-3. Moreover, KPC-Kp strains carrying a mutated blaKPC-3 gene exhibited high MIC values (p value < 0.001) compared to wild-type blaKPC-3. In conclusion, the DD test resulted as a valid alternative to the BMD method to determine the in vitro susceptibility to CFD, while the strip test exhibited major limitations.

Objectives: Implementation of standard laboratory practices towards accurate antimicrobial susceptibility testing (AST) is challenging in resource-constrained settings. Efforts to improve AST are required to address knowledge and practice gaps in such settings. In this study, we aimed to address these gaps through external quality assurance surveys and mentoring of laboratories in Pakistan. Methods: This prospective study (May 2017–September 2019) included 10 consenting laboratories. External quality assessment (EQA) was conducted quarterly and performance scored. Each EQA cycle was followed by an on-site technical visit during which AST methodology, quality procedures and laboratory safety were evaluated using a questionnaire developed for this study. Cumulative scores of performance in the EQA and in the technical evaluation were designated "Composite Laboratory Performance Score; CLPS". During on-site visits, feedback provided was to each participating laboratory towards addressing gaps identified. Results: Over the course of the study, our data show significant improvement in CLPS amongst the laboratories included. While improvement in the CLPS scores varied between laboratories, a linear regression model showed improvement within the cohort from 21.37 (May 2017) to 91.5 (September 2019); a significant overall increase of 70.13 points (p = 0.001). Conclusion: Interventions to improve AMR surveillance include quality assured reporting of antimicrobial resistance. Our data show that in resource-limited settings EQA surveys and on-site evaluations followed by guidance contribute towards such improvement. We propose that this model would be a useful tool for laboratory strengthening in such settings. [ABSTRACT FROM AUTHOR]

Biofilm-associated bacteria exhibit profound changes in bacterial physiology. They thrive in the environment but also in the human host in protected sessile communities. Antimicrobial therapy usually fails, despite the absence of genotypic resistance, and it is commonly accepted that biofilm-grown bacteria are up to 1,000-fold more resistant than planktonic cells. We are only at the beginning to understand the reasons for biofilm recalcitrance, and systematic approaches to describe biofilm-induced tolerance phenotypes are lacking. In this study, we investigated a large and highly diverse collection of 352 clinical Pseudomonas aeruginosa isolates for their antimicrobial susceptibility profiles under biofilm growth conditions towards the antibiotics ciprofloxacin, tobramycin, and colistin. We discovered characteristic patterns of drug-specific killing activity and detected conditional tolerance levels far lower (in the range of the minimal inhibitory concentration (MIC)), but also far higher (up to 16,000-fold increase compared to planktonic cells) than generally believed. This extremely broad distribution of biofilm-induced tolerance phenotypes across the clinical isolates was greatly influenced by the choice of the antibiotic. We furthermore describe cross-tolerance against ciprofloxacin and tobramycin, but not colistin, and observed an additive activity between biofilm-induced tolerance and genetically determined resistance. This became less evident when the biofilm-grown cells were exposed to very high antibiotic concentrations. Although much more remains to be learned on the molecular mechanisms underlying biofilm-induced tolerance, our data on intra-species variations in tolerance profiles provide valuable new insights. Furthermore, our observation that colistin appears to act independently of the tolerance mechanisms of individual clinical strains could make colistin a valuable therapeutic option in chronic biofilm-associated infections characterized by the presence of particularly tolerant strains. [ABSTRACT FROM AUTHOR]

Biofilm-associated bacteria exhibit profound changes in bacterial physiology. They thrive in the environment but also in the human host in protected sessile communities. Antimicrobial therapy usually fails, despite the absence of genotypic resistance, and it is commonly accepted that biofilm-grown bacteria are up to 1,000-fold more resistant than planktonic cells. We are only at the beginning to understand the reasons for biofilm recalcitrance, and systematic approaches to describe biofilm-induced tolerance phenotypes are lacking. In this study, we investigated a large and highly diverse collection of 352 clinical Pseudomonas aeruginosa isolates for their antimicrobial susceptibility profiles under biofilm growth conditions towards the antibiotics ciprofloxacin, tobramycin, and colistin. We discovered characteristic patterns of drug-specific killing activity and detected conditional tolerance levels far lower (in the range of the minimal inhibitory concentration (MIC)), but also far higher (up to 16,000-fold increase compared to planktonic cells) than generally believed. This extremely broad distribution of biofilm-induced tolerance phenotypes across the clinical isolates was greatly influenced by the choice of the antibiotic. We furthermore describe cross-tolerance against ciprofloxacin and tobramycin, but not colistin, and observed an additive activity between biofilm-induced tolerance and genetically determined resistance. This became less evident when the biofilm-grown cells were exposed to very high antibiotic concentrations. Although much more remains to be learned on the molecular mechanisms underlying biofilm-induced tolerance, our data on intra-species variations in tolerance profiles provide valuable new insights. Furthermore, our observation that colistin appears to act independently of the tolerance mechanisms of individual clinical strains could make colistin a valuable therapeutic option in chronic biofilm-associated infections characterized by the presence of particularly tolerant strains.

Drug-resistant bacterial strains seriously threaten human health. Rapid screening of antibiotics is urgently required to improve clinical treatment. Conventional methods of antimicrobial susceptibility testing rely on turbidimetry that is evident only after several days of incubation. The lengthy time of the assay can delay clinical treatment. Here, we proposed a single-cell level rapid system based on a microfluidic chip. The detection period of 30 min to 2 h was significantly shorter than the conventional turbidity-based method. To promote detection efficiency, 16 independent channels were designed, permitting the simultaneous screening of 16 drugs in the microfluidic chip. Prepositioning of drugs in the chip permitted prolonged transportation and storage. This may allow for the widespread use of the novel system, particularly in the regions where medical facilities are scarce. The growth curves were reported rapidly through a custom code in Matlab after tracking and photographing the bacteria during microscopy examination. The capability of the proposed system was validated by antimicrobial susceptibility testing trials with standard strains. The system provides a potentially useful detection tool for drug-resistant bacteria.

External Quality Assessment schemes (EQAS) are mandatory to ensure quality standards in diagnostic methods and achieve laboratory accreditation. As host institution for two German culture-based bacteriology EQAS (RV-A and RV-B), we investigated the obtained data of 590 up to 720 surveys per year in RV-A and 2,151 up to 2,929 in RV-B from 2006 to 2023. As educational instruments, they function to review applied methodology and are valuable to check for systemic- or method-dependent failures in microbiology diagnostics or guidelines. Especially, containment of multi-resistant bacteria in times of rising antibiotic resistance is one major point to assure public health. The correct identification and reporting of these strains is therefore of high importance to achieve this goal. Moreover, correct antimicrobial susceptibility testing (AST) per se is important for selecting appropriate therapy, to restrict broad-spectrum antibiotics and minimize resistance development. The reports of participating laboratories displayed a high level of correct identification results in both schemes with mostly consistent failure rates around 2.2% (RV-A) and 3.9% (RV-B) on average. In contrast, results in AST revealed increasing failure rates upon modification of AST requirements concerning adherence to standards and subsequent bacterial species-specific evaluation. Stratification on these periods revealed in RV-A a moderate increase from 1.3% to 4.5%, while in RV-B failure rates reached 14% coming from 4.3% on average. Although not mandatory, subsequent AST evaluation and consistent reporting are areas of improvement to benefit public health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lindenberg, Waldmann, Suerbaum, Schlüter and Ziesing.)

Antimicrobial susceptibility testing (AST) should be conducted in a standardized manner prior to the start of an antimicrobial treatment. For fastidious bacteria, such as porcine Mycoplasma (‘Mesomycoplasma’) spp., specifically M. hyorhinis, neither guidelines or standards for the performance of AST, nor quality control strains for the validation of AST results are approved by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CLSI- and EUCAST-approved quality control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were chosen to validate AST by broth microdilution using modified Friis broth, developed as growth medium for porcine Mycoplasma (‘Mesomycoplasma’) spp. The antimicrobial agents doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin were examined using customized SensititreTM microtiter plates. Minimal inhibitory concentrations, determined after 24, 48, and 72 h, were mostly within the CLSI-approved quality control ranges for defined antimicrobial agents. We propose the use of the combination of E. faecalis ATCC 29212 and S. aureus ATCC 29213 as surrogate quality control strains for the validation of future AST results obtained for M. hyorhinis by broth microdilution using modified Friis broth. [ABSTRACT FROM AUTHOR]

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) comprises a global threat to humans and animals. Here, we report and characterize the MRSA t304/ST6 variant which, to our knowledge, represents the first case found in bovine clinical mastitis. In general, the MRSA t304/ST6 variant is rarely described in livestock, contrary to humans where it is widely recognized. Phenotypic and genotypic resistance profiling showed that the bovine-MRSA t304/ST6 isolate expressed low susceptibility toward cefoxitin (MICcefoxitin = 16 µg/mL) and carried the mecA resistance gene in the SCCmec IVa. The bovine-MRSA t304/ST6 isolate carried a plasmid similar to that which has been frequently observed among human-MRSA t304/ST6 isolates in Denmark (GenBank accession no. NZ_CP047022). In addition, a Staphylococcus prophage 3 (ϕSA3) was detected, encoding an immune evasion cluster (IEC) of putative virulence genes associated with human host-specificity (sea, sak, and scn). Taken together, these findings suggest that the MRSA t304/ST6 found in this study represents a recent host-jump event, with human to cow transmission. This study emphasizes the importance of and the need for performance of antimicrobial resistance surveillance among bovine mastitis pathogens, including S. aureus and MRSA.

Introduction. Non-tuberculous Mycobacteria (NTM) is a group of mycobacteria distinct from the Mycobacterium tuberculosis complex. They can cause opportunistic infections, especially in immunocompromised individuals. Gap Statement. Over the last few years, there has been a growing concern regarding the distribution and antimicrobial resistance of NTM in Malaysia. however, a comprehensive study to fully grasp the NTM situation has yet to be conducted. Aim. This study aimed to investigate the species distribution and antimicrobial susceptibility patterns of NTM isolated from clinical samples in Malaysia from 2018 to 2022. Methodology. A retrospective analysis was conducted on NTM isolates obtained from various clinical specimens over a span of five years. The isolates were identified using phenotypic and molecular techniques, and antimicrobial susceptibility profiles for clinically significant isolates were determined using minimum inhibitory concentration. Results. The study revealed a diverse distribution of NTM species in Malaysia, with Mycobacteroides abscessus complex and Mycobacterium avium complex emerging as the most predominant. Furthermore, the antimicrobial susceptibility patterns showed varying degrees of resistance to commonly used antibiotics, highlighting the significance of treatment tailored to susceptibility testing results. Conclusion. This study provides valuable perspective into the epidemiology of NTM in Malaysia. The information gained from this study should prove useful for empirically treating serious NTM infections prior to species identification and the availability of antimicrobial susceptibility testing results.

Background: Accelerating the diagnosis of bacteremia is one of the biggest challenges in clinical microbiology departments. The fast establishment of a correct treatment is determinant on bacteremic patients’ outcomes. Our objective was to evaluate the impact of antimicrobial therapy and clinical outcomes of a rapid blood culture workflow protocol in positive blood cultures with Gram-negative bacilli (GNB). Methods: A quasi-experimental before–after study was performed with two groups: (i) control group (conventional work-protocol) and (ii) intervention group (rapid workflow-protocol: rapid identification by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) and antimicrobial susceptibility testing (AST) from bacterial pellet without overnight incubation). Patients were divided into different categories according to the type of intervention over treatment. Outcomes were compared between both groups. Results: A total of 313 patients with GNB-bacteremia were included: 125 patients in the control group and 188 in the intervention. The time from positive blood culture to intervention on antibiotic treatment decreased from 2.0 days in the control group to 1.0 in the intervention group (p < 0.001). On the maintenance of correct empirical treatment, the control group reported 2.0 median days until the clinical decision, while in the intervention group was 1.0 (p < 0.001). In the case of treatment de-escalation, a significant difference between both groups (4.0 vs. 2.0, p < 0.001) was found. A decreasing trend on the change from inappropriate treatments to appropriate ones was observed: 3.5 vs. 1.5; p = 0.12. No significant differences were found between both groups on 7-days mortality or on readmissions in the first 30-days. Conclusions: Routine implementation of a rapid workflow protocol anticipates the report of antimicrobial susceptibility testing results in patients with GNB-bacteremia, decreasing the time to effective and optimal antibiotic therapy.

Salmonella, one of the most common foodborne pathogens, poses a serious threat to human health. In recent years, the antibiotic resistance of Salmonella has become serious as well, which has strengthened the harm of Salmonella to human health. In this paper, a simple and effective electrochemical approach was developed to obtain profile of the antimicrobial susceptibility. Screen-printed carbon electrodes (SPCEs) were used to detect various concentrations of target bacteria at the same time, which have good stability, low cost, and easy mass production. The electroactive redox, resazurin, was used to monitor levels of metabolically active bacteria. As a demonstration, the antibacterial effect of ofloxacin and penicillin on Salmonella gallinarum (S. gallinarum) isolates was evaluated, and the minimum inhibitory concentration (MIC) was measured as well. In the real sample measurement, the MIC obtained was similar to that obtained by conventional antimicrobial susceptibility testing (AST). In this assay, bacterial activity was quantified sensitively and accurately, and the detection time was greatly reduced compared with conventional AST (16–20 h), which means that metabolic capacity of live bacteria could be observed after 1 h of incubation. We were able to clearly detect bacteria above 102 CFU/m. This method aims to nonspecific and can be widely applied to the detection of a variety of drug-resistant bacteria, providing an experimental basis for the rational use of antibiotics and bacterial resistance mechanisms. [ABSTRACT FROM AUTHOR]

The abuse of antibiotics has become a global public safety issue, leading to the development of antimicrobial resistance (AMR). The development of antimicrobial susceptibility testing (AST) is crucial in reducing the growth of AMR. However, traditional AST methods are time-consuming (e.g., 24–72 h), labor-intensive, and costly. Here, we propose a controlled-diffusion centrifugal microfluidic platform (CCM) for rapid AST to obtain highly precise minimum inhibitory concentration (MIC) values. Antibiotic concentration gradients are generated by controlled moving and diffusing of antibiotic and buffer solution along the main microchannel within 3 min. The solution and bacterial suspension are then injected into the outermost reaction chamber by simple centrifugation. The CCM successfully determined the MIC for three commonly used antibiotics in clinical settings within 4–9 h. To further enhance practicality, reduce costs, and meet point-of-care testing demands, we have developed an integrated mobile detection platform for automated MIC value acquisition. The proposed CCM is a simple, low-cost, and portable method for rapid AST with broad clinical and in vitro applications. [Display omitted] • A controlled-diffusion centrifugal microfluidic for rapid AST was successfully proposed. • Antibiotic concentration gradients generated by combined diffusion and centrifugation. • An integrated mobile detection platform to automatically obtain MIC values. [ABSTRACT FROM AUTHOR]

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed “bicarbonate (NaHCO3)-responsiveness” was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro “susceptibility” to standard β-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these β-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller–Hinton Broth (MHB); and 15 MIC-resistant to both β-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains.

Antimicrobial susceptibility testing (AST) should be conducted in a standardized manner prior to the start of an antimicrobial treatment. For fastidious bacteria, such as porcine Mycoplasma (‘Mesomycoplasma’) spp., specifically M. hyorhinis, neither guidelines or standards for the performance of AST, nor quality control strains for the validation of AST results are approved by organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CLSI- and EUCAST-approved quality control strains Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were chosen to validate AST by broth microdilution using modified Friis broth, developed as growth medium for porcine Mycoplasma (‘Mesomycoplasma’) spp. The antimicrobial agents doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin were examined using customized SensititreTM microtiter plates. Minimal inhibitory concentrations, determined after 24, 48, and 72 h, were mostly within the CLSI-approved quality control ranges for defined antimicrobial agents. We propose the use of the combination of E. faecalis ATCC 29212 and S. aureus ATCC 29213 as surrogate quality control strains for the validation of future AST results obtained for M. hyorhinis by broth microdilution using modified Friis broth.

The emergence and prevalence of antibiotic-resistant bacteria are an increasing cause of death worldwide, resulting in a global ‘call to action’ to avoid receding into an era lacking effective antibiotics. Despite the urgency, the healthcare industry still relies on a single in vitro bioassay to determine antibiotic efficacy. This assay fails to incorporate environmental factors normally present during host-pathogen interactions in vivo that significantly impact antibiotic efficacy. Here we report that standard antimicrobial susceptibility testing (AST) failed to detect antibiotics that are in fact effective in vivo; and frequently identified antibiotics that were instead ineffective as further confirmed in mouse models of infection and sepsis. Notably, AST performed in media mimicking host environments succeeded in identifying specific antibiotics that were effective in bacterial clearance and host survival, even though these same antibiotics failed in results using standard test media. Similarly, our revised media further identified antibiotics that were ineffective in vivo despite passing the AST standard for clinical use. Supplementation of AST medium with sodium bicarbonate, an abundant in vivo molecule that stimulates global changes in bacterial structure and gene expression, was found to be an important factor improving the predictive value of AST in the assignment of appropriate therapy. These findings have the potential to improve the means by which antibiotics are developed, tested, and prescribed.

Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sepsis is a life-threatening condition with systemic inflammatory responses caused by bacterial infections. Considering the emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), sepsis is a great threat to public health. The gold standard methods for antimicrobial susceptibility testing (AST), however, take at least approximately 3 days to implement the entire blood culture, pure culture, and AST processes. To overcome the time-consuming nature of conventional AST, a method employing a chromatic biosensor composed of poly(diacetylene), alginate, and LB broth (PAL) is introduced in this study. Compared to the gold standards, AST with PAL biosensors can be completed within a time frame as short as 16 h. Such a significant reduction in time is possible because the consecutive cultures and AST are carried out simultaneously by encapsulating the bacterial nutrients and detection molecules into a single component. The bead-like hydrogel sensors were used in their freeze-dried form, which endows them with portability and stability, thus making them adequate for point-of-care testing. The PAL biosensor yields minimum inhibitory concentrations comparable to those from the Clinical and Laboratory Standards Institute, and the applicability of the biosensor is further shown in MRSA-infected mice.

Introduction:Antimicrobial susceptibility testing is key in modern clinical microbiology. With pandemic emergence of (multi-)antibiotic resistance, methods to detect and quantify resistance of clinically important bacterial species are imperative. Historically, antimicrobial susceptibility testing (AST) was mostly performed using methods relying on bacterial growth. Such methods may be time-consuming and more rapid alternatives have been actively sought for. Areas covered:Among the new AST methods there are many that focus on detection of causal resistance genes and/or gene mutations. The approaches most used are based on nucleic acid amplification and, more recently, high-throughput (next generation) sequencing of amplified targets and complete microbial genomes. The authors provide a review of PCR-mediated and genomic AST methods used for human and veterinary pathogens and show where these approaches work well or may become difficult to interpret. Expert commentary:Microbial genome sequencing will play an important role in the field of AST, but there remain issues to be resolved. These include the development of user friendly data analysis, reducing the duration and cost of sequencing and comprehensiveness of the databases. In addition, clinical evaluation studies need to be performed involving real-life patients. [ABSTRACT FROM PUBLISHER]

The objective of the study was to explore antimicrobial resistance gene determinant, and phenotypic antibiotic susceptibility, data for Fusobacterium necrophorum from a collection of UK strains. Antimicrobial resistance genes detected in publicly available assembled whole genome sequences were investigated for comparison. Three hundred and eighty five F. necrophorum strains (1982–2019) were revived from cryovials (Prolab). Subsequent to sequencing (Illumina) and quality checking, 374 whole genomes were available for analysis. Genomes were interrogated, using BioNumerics (bioMérieux; v 8.1), for the presence of known antimicrobial resistance genes (ARGs). Agar dilution susceptibility results for 313 F. necrophorum isolates (2016–2021) were also examined. The phenotypic data for the 313 contemporary strains demonstrated potential resistance to penicillin in three isolates, using EUCAST v 11.0 breakpoints, and 73 (23%) strains using v 13.0 analysis. All strains were susceptible to multiple agents using v 11.0 guidance other than clindamycin (n = 2). Employing v 13.0 breakpoints, metronidazole (n = 3) and meropenem (n = 13) resistance were also detected. The tet(O) , tet(M) , tet(40) , aph(3')-III , ant(6)-la and bla OXA-85 ARGs were present in publicly available genomes. tet(M) , tet(32), erm(A) and erm(B) were found within the UK strains, with correspondingly raised clindamycin and tetracycline minimum inhibitory concentrations. Susceptibility to antibiotics recommended for the treatment of F. necrophorum infections should not be assumed. With evidence of potential ARG transmission from oral bacteria, and the detection of a transposon-mediated beta-lactamase resistance determinant in F. necrophorum , surveillance of both phenotypic and genotypic antimicrobial susceptibility trends must continue, and increase. • Resistance to several antibiotics was demonstrated. • All strains were susceptible to piperacillin/tazobactam. • Multiple antimicrobial resistance genes were detected in publicly available genomes. • tet(M), tet(32), erm(A) and erm(B) were discovered in sequenced clinical strains. • Raised minimum inhibitory concentrations aligned with resistance gene detection. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Introduction: Mycobacterium abscessus is an emerging mycobacteria that is responsible for lung diseases and healthcare-associated extrapulmonary infections. Recent findings support its taxonomic status as a single species comprising 3 subspecies designated abscessus, bolletii and massiliense. We performed a review of English-language publications investigating all three of these subspecies. Areas covered: Worldwide, human infections are often attributable to environmental contamination, although the isolation of M. abscessus in this reservoir is very rare. Basic research has demonstrated an association between virulence and cell wall components and cording, and genome analysis has identified gene transfer from other bacteria. The bacteriological diagnosis of M. abscessus is based on innovative tools combining molecular biology and mass spectrometry. Genotypic and phenotypic susceptibility testing are required to predict the success of macrolide (clarithromycin or azithromycin)-based therapeutic regimens. Genotyping methods are helpful to assess relapse and cross-transmission and to search for a common source. Treatment is not standardised, and outcomes are often unsatisfactory. Expert commentary: M. abscessus is still an open field in terms of clinical and bacteriological research. Further knowledge of its ecology and transmission routes, as well as host-pathogen interactions, is required. Because the number of human cases is increasing, it is also necessary to identify more active treatments and perform clinical trials to assess standard effective regimens. [ABSTRACT FROM AUTHOR]

Background: The Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines are the most popular breakpoint guidelines used in antimicrobial susceptibility testing worldwide. The EUCAST guidelines are freely available to users while CLSI is available for non-members as a package of three documents for US $500 annually. This is prohibitive for clinical microbiology laboratories in resource poor settings. We set out to compare antibiotic susceptibility determined by the two guidelines to determine whether adoption of EUCAST guidelines would significantly affect our susceptibility patterns. Methods: We reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) isolates from an automated microbiology identification system (VITEK-2) at the Aga Khan University Hospital Nairobi's Pathology department. These MICs were then analyzed using both CLSI 2015 and EUCAST 2015 guidelines and classified as resistant, intermediate or susceptible. We compared the susceptibility and agreement between the CLSI and EUCAST categorizations. Results: Susceptibility data from a total of 5165 E. coli, 1103 S. aureus and 532 P. aeruginosa isolates were included. The concordance rates of the two guidelines for E. coli, S. aureus and P. aeruginosa ranged from 78.2 to 100 %, 94.6 to 100 % and 89.1 to 95.5 % respectively. The kappa statistics for E. coli MICs revealed perfect agreement between CLSI and EUCAST for cefotaxime, ceftriaxone and trimethoprim-sulfamethoxazole, almost perfect agreement for ampicillin, ciprofloxacin, cefuroxime, gentamicin and ceftazidime, substantial agreement for meropenem, moderate agreement for cefepime and amoxicillin-clavulanate, fair agreement for nitrofurantoin and poor agreement for amikacin. For S. aureus the kappa statistics revealed perfect agreement for penicillin, trimethoprim-sulfamethoxazole, levofloxacin, oxacillin, linezolid and vancomycin, almost perfect agreement for clindamycin, erythromycin and tetracycline and moderate agreement for gentamicin. For P. aeruginosa the kappa analysis revealed moderate to almost perfect agreement for all the anti-pseudomonal antibiotics. Conclusion: The results show comparable antibiotic susceptibility patterns between CLSI and EUCAST breakpoints. Given that EUCAST guidelines are freely available, it makes it easier for laboratories in resource poor settings to have an updated and readily available reference for interpreting antibiotic susceptibilities. [ABSTRACT FROM AUTHOR]

Drug-resistant bacterial strains seriously threaten human health. Rapid screening of antibiotics is urgently required to improve clinical treatment. Conventional methods of antimicrobial susceptibility testing rely on turbidimetry that is evident only after several days of incubation. The lengthy time of the assay can delay clinical treatment. Here, we proposed a single-cell level rapid system based on a microfluidic chip. The detection period of 30 min to 2 h was significantly shorter than the conventional turbidity-based method. To promote detection efficiency, 16 independent channels were designed, permitting the simultaneous screening of 16 drugs in the microfluidic chip. Prepositioning of drugs in the chip permitted prolonged transportation and storage. This may allow for the widespread use of the novel system, particularly in the regions where medical facilities are scarce. The growth curves were reported rapidly through a custom code in Matlab after tracking and photographing the bacteria during microscopy examination. The capability of the proposed system was validated by antimicrobial susceptibility testing trials with standard strains. The system provides a potentially useful detection tool for drug-resistant bacteria.