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Introduction. The alarming rise in urinary tract infection (UTI) antimicrobial resistance has resulted from a combination of high prevalence, low specificity and the lack of a rapid, point-of-care (POC) antibiotic susceptibility test (AST), which has led to the overuse/inappropriate use of antibiotics.Aim. This study aimed to evaluate the performance of a rapid POC phenotypic AST device in reporting susceptibility information within 2 h.Methodology. Instrument calibration was performed with model bacteria and fluorescent microbeads to determine the dynamic range and limit of detection for quantifying concentrations of bacteria and demonstrate the ability to rapidly differentiate susceptible and resistant model bacteria. We then evaluated 30 presumptive UTI-positive patient urine samples in a clinical pilot study using a panel of 5 common UTI antibiotics plus a growth control and compared our results to the hospital standard of care AST.Results. Our device was able to robustly detect and quantify bacteria concentrations from 50 to 105 colony-forming units (c.f.u.) ml-1. The high sensitivity of this measurement technique enabled the device to differentiate between susceptible and resistant model bacteria with 100 % specificity over a 2 h growth period. In the clinical pilot study, an overall categorical agreement (CA) of 90.7 % was observed (sensitivity=91.4 %, specificity=88.9 %, n=97) with performance for individual drugs ranging from 85 % CA (ceftazidime) to 100 % (nitrofurantoin).Conclusions. By reducing the typical timeframe for susceptibility testing from 2-3 days to 2 h, our POC phenotypic AST can provide critical information to clinicians prior to the administration of antibiotic therapy.

Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment. Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms. Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding. Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role. Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB , including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases. Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Colistin resistance testing methods such as broth microdilution (BMD) are time-consuming and labour intensive for clinical laboratories. MBT Lipid Xtract Kit on MALDI Biotyper Sirius System (Bruker, Billerica, MA, USA) utilizes lipidomic analysis to identify specific cell wall modifications associated with colistin resistance. We compared MBT to BMD (ComASP Colistin, Liofilchem) across 36 Gram-negative isolates (non-resistant MIC ≤2 µg ml -1 , resistant MIC ≥4 µg ml -1 ). All samples were tested twice on MBT with discrepant results repeated before assessing categorical agreement between MBT and BMD. 44.4% (16/36) of isolates were colistin resistant via BMD. MBT Lipid Xtract had 80.6% agreement (29/36) with BMD, with 5/7 discrepancies corrected to match upon repeat testing. There was 100% agreement for Escherichia coli isolates ( n =16). The whole-genome sequencing was completed on the two discrepant Klebsiella pneumoniae isolates, with variants within colistin resistance-associated loci identified (MIC 0.5 µg ml -1 : arnC S30T, pmrB T246A, lapB N212T, lpxM S253G, crrB Q287K and MIC >16 µg ml -1 : arnC S30T, pmrB R90insRN, pmrB T246A, pmrA E57G, lpxM S253G). Further evaluation, particularly for non- E. coli , of MBT is required prior to implementation in clinical laboratories.

In 2020 and 2022, nine cases of surgical site infections with a methicillin-resistant Staphylococcus aureus (MRSA) were diagnosed in horses in an equine referral clinic. Sixteen isolates (horses, n =9; environment, n =3; and staff members, n =4) were analysed retrospectively using Nanopore whole-genome sequencing to investigate the relatedness of two suspected MRSA outbreaks (2020 and 2022). The MRSA isolates belonged to ST398 and ST612. ST398 genomes from 2020 and 2022 formed three phylogenetic clusters. The first ST398 cluster from 2020 consisted of isolates from five horses and one staff member, and we suspected within clinic transmission. The second cluster of ST398 isolates from 2022 originated from two horses and two staff members but showed higher single nucleotide polymorphism (SNP) distances. One ST398 isolate from an individual staff member was not related to the other two clusters. The ST612 isolates were isolated in 2022 from two horses and three environmental samples and showed very low SNP distances (<7 SNPs), indicating the transmission of MRSA ST612 in this clinic in 2022. Molecular characterization revealed an abundant set of virulence genes and plasmids in the ST612 isolates in comparison to ST398 isolates. Phenotypic antimicrobial susceptibility showed that differences between the two sequence types were consistent with the genetic characteristics. MRSA ST612 has not been reported in Europe before, but it is a dominant clone in African hospitals and has been described in horses and people working with horses in Australia, indicating the importance of surveillance.

Introduction . The global spread of Acinetobacter spp., particularly the Acinetobacter calcoaceticusbaumannii (ACB) complex, has led to its recognition as a significant pathogen by the World Health Organization (WHO). The increasing resistance of the ACB complex to multiple antibiotics presents a challenge for treatment, necessitating accurate antibiotic susceptibility profiling after isolation. Hypothesis or gap statement . There is limited understanding of the antimicrobial resistance and chlorhexidine, a biocide, susceptibility profiles of ACB complex strains, especially in clinical settings in Turkey. Aim . This study aimed to identify ACB complex strains recovered from various clinical specimens at Hacettepe University Hospitals in Ankara, Turkey, in 2019, and to assess identification, their antibiotic and chlorhexidine susceptibility profiles, and genomic relatedness. Methodology . Eighty-two ACB complex strains were identified using MALDI-TOF MS. Susceptibility testing to 12 antibiotics was conducted using the disc diffusion method, and colistin, chlorhexidine susceptibility was assessed using the broth microdilution technique, following the latest EUCAST and CLSI guidelines. ACB complex members with reduced chlorhexidine sensitivity were further analyzed by pulsed-field gel electrophoresis (PFGE) for bacterial typing. Results . Among the isolates, 1.2% were multidrug-resistant (MDR), 73.2% were extensively drug-resistant (XDR), and 12.2% were pandrug-resistant (PDR). Carbapenem resistance was found in 86.7% of MDR, PDR, and XDR strains. Colistin resistance was observed in 15.8% of isolates, and 18.2% exhibited decreased susceptibility to chlorhexidine. PFGE revealed seven different clones among strains with reduced chlorhexidine sensitivity, indicating vertical transmission within the hospital. Conclusion . This study highlights the reduced susceptibility to chlorhexidine in ACB complex members and provides epidemiological insights into their spread. The findings underscore the importance of screening for antimicrobial resistance and biocide susceptibility profiles to effectively manage healthcare-associated infections.

Introduction. Klebsiella spp. are important bacteria that colonize the human intestine, especially in preterm infants; they can induce local and systemic disease under specific circumstances, including inflammatory bowel disease, necrotizing enterocolitis and colorectal cancer. Hypothesis. Klebsiella spp. colonized in the intestine of the neonates in the neonatal intensive care unit (NICU) may be associated with disease and antibiotic resistance, which will be hazardous to the children. Aim. Our aim was to know about the prevalence, antimicrobial resistance and genome characteristics of Klebsiella spp. in neonate carriers. Methodology. Genome sequencing and analysis, and antimicrobial susceptibility testing were mainly performed in this study. Results. The isolation rates of Klebsiella spp. strains were 3.7% (16/436) in 2014 and 4.3% (18/420) in 2021. Cases with intestinal-colonized Klebsiella spp. were mainly infants with low birth weights or those with pneumonia or hyperbilirubinemia. According to the core-pan genomic analysis, 34 stains showed gene polymorphism and a sequence type (ST) of an emerging high-risk clone (ST11). Eight strains (23.5%) were found to be resistant to 2 or more antibiotics, and 46 genes/gene families along with nine plasmids were identified that conferred resistance to antibiotics. In particular, the two strains were multidrug-resistant. Strain A1256 that is related to Klebsiella quasipneumoniae subsp. similipneumoniae was uncommon, carrying two plasmids similar to IncFII and IncX3 that included five antibiotic resistance genes. Conclusion. The prevention and control of neonatal Klebsiella spp. colonization in the NICU should be strengthened by paying increased attention to preventing antimicrobial resistance in neonates.

Introduction. Sporotrichosis is a subcutaneous infection caused by dimorphic Sporothrix species embedded in the clinical clade. Fungi have virulence factors, such as biofilm and melanin production, which contribute to their survival and are related to the increase in the number of cases of therapeutic failure, making it necessary to search for new options. Gap statement. Proton pump inhibitors (PPIs) have already been shown to inhibit the growth and melanogenesis of other fungi. Aim. Therefore, this study aimed to evaluate the effect of the PPIs omeprazole (OMP), rabeprazole (RBP), esomeprazole, pantoprazole and lansoprazole on the susceptibility and melanogenesis of Sporothrix species, and their interactions with itraconazole, terbinafine and amphotericin B. Methodology. The antifungal activity of PPIs was evaluated using the microdilution method, and the combination of PPIs with itraconazole, terbinafine and amphotericin B was assessed using the checkerboard method. The assessment of melanogenesis inhibition was assessed using grey scale. Results. The OMP and RBP showed significant MIC results ranging from 32 to 256 µg ml -1 and 32 to 128 µg ml -1 , respectively. Biofilms were sensitive, with a significant reduction ( P <0.05) in metabolic activity of 52% for OMP and 50% for RBP at a concentration of 512 µg ml -1 and of biomass by 53% for OMP and 51% for RBP at concentrations of 512 µg ml -1 . As for the inhibition of melanogenesis, only OMP showed inhibition, with a 54% reduction. Conclusion. It concludes that the PPIs OMP and RBP have antifungal activity in vitro against planktonic cells and biofilms of Sporothrix species and that, in addition, OMP can inhibit the melanization process in Sporothrix species.

Introduction. Pre-existing fluoroquinolones (FQs) resistance is a major threat in treating multidrug-resistant (MDR) tuberculosis. Sitafloxacin (Sfx) is a new broad-spectrum FQ. Hypothesis. Sfx is more active against drug-resistant Mycobacterium tuberculosis (Mtb) isolates. Aim. To determine whether there is cross-resistance between Sfx and ofloxacin (Ofx), levofloxacin (Lfx) and moxifloxacin (Mfx) in MDR Mtb. Methods. A total of 106 clinical Mtb isolates, including 23 pan-susceptible and 83 MDR strains, were analysed for Sfx, Lfx and Mfx resistance using MIC assay. The isolates were also subjected to whole-genome sequencing to analyse drug-resistant genes. Results. Sfx exhibited the most robust inhibition activity against Mtb clinical isolates, with a MIC 50 of 0.0313 µg ml -1 and MIC 90 of 0.125 µg ml -1 , which was lower than that of Mfx (MIC 50 = 0.0625 µg ml -1 , MIC 90 = 1 µg ml -1 ) and Lfx (MIC 50 = 0.125 µg ml -1 , MIC 90 = 2 µg ml -1 ). We determined the tentative epidemiological cut-off values as 0.5 µg ml -1 for Sfx. Also, 8.43% (7/83), 43.37% (36/83), 42.17% (35/83) and 51.81% (43/83) MDR strains were resistant to Sfx, Mfx, Lfx and Ofx, respectively. Cross-resistance between Ofx, Lfx and Mfx was 80.43% (37/46). Only 15.22% (7/46) of the pre-existing FQs resistance isolates were resistant to Sfx. Among the 30 isolates with mutations in gyrA or gyrB , 5 (16.67%) were Sfx resistant. The combination of Sfx and rifampicin could exert partial synergistic effects, and no antagonism between Sfx and six clinically important anti-Mtb antibiotics was evident. Conclusion. Sfx exhibited superior activity against MDR isolates comparing to Lfx and Mfx, and could potentially overcome the majority pre-existing FQs resistance in Mtb strains.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections. Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed. Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity. Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells. Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus . Two compounds were moderately cytotoxic, and the rest were highly cytotoxic. Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Introduction. Maternal screening tests and prophylactic antibiotics are important to prevent neonatal and infant group B streptococcal (GBS) infections. Hypothesis/Gap Statement. The performance of enrichment broth media for GBS screening that are available in Japan is unclear. Whole-genome data of GBS isolates from pregnant women in Japan is lacking. Aim. The aim of this study was to compare the protocol performance of six enrichment broths and two subculture agar plates, which were all available in Japan, for GBS detection. In addition, we showed whole-genome data of GBS isolates from pregnant women in Japan. Methodology. We collected 133 vaginal-rectal swabs from pregnant women visiting clinics and hospitals in Nagasaki Prefecture, Japan, and compared the protocol performance of 6 enrichment broths and 2 subculture agar plates. All GBS isolates collected in this study were subjected to whole-genome sequencing analysis. Results. We obtained 133 vaginal-rectal swabs from pregnant women at 35-37 weeks of gestation from 8 private clinics and 2 local municipal hospitals within Nagasaki Prefecture, Japan. The detection rate of the protocol involving the six enrichment broths and subsequent subcultures varied between 95.5 and 100 %, depending on the specific choice of enrichment broth. The GBS carriage rate among pregnant women in this region was 18.8 %. All 25 isolates derived from the swabs were susceptible to penicillin, whereas 48 and 36 % of the isolates demonstrated resistance to erythromycin and clindamycin, respectively. The distribution of serotypes was highly diverse, encompassing seven distinct serotypes among the isolates, with the predominant serotype being serotype V ( n = 8). Serotype V isolates displayed a tendency towards increased resistance to erythromycin and clindamycin, with all resistant isolates containing the ermB gene. Conclusion. There was no difference in performance among the culture protocols evaluated in this study. GBS strains isolated from pregnant women appeared to have greater genomic diversity than GBS strains detected in neonates/infants with invasive GBS infections. To confirm this result, further studies with larger sample sizes are needed.

Introduction. The development of new antifungal drugs has become a global priority, given the increasing cases of fungal diseases together with the rising resistance to available antifungal drugs. In this scenario, drug repositioning has emerged as an alternative for such development, with advantages such as reduced research time and costs. Gap statement. Propafenone is an antiarrhythmic drug whose antifungal activity is poorly described, being a good candidate for further study. Aim. This study aims to evaluate propafenone activity against different species of Candida spp. to evaluate its combination with standard antifungals, as well as its possible action mechanism. Methodology. To this end, we carried out tests against strains of Candida albicans , Candida auris , Candida parapsilosis , Candida tropicalis , Candida glabrata and Candida krusei based on the evaluation of the MIC, minimum fungicidal concentration and tolerance level, along with checkerboard and flow cytometry tests with clinical strains and cell structure analysis by scanning electron microscopy (SEM). Results. The results showed that propafenone has a 50% MIC ranging from 32 to 256 µg ml -1 , with fungicidal activity and positive interactions with itraconazole in 83.3% of the strains evaluated. The effects of the treatments observed by SEM were extensive damage to the cell structure, while flow cytometry revealed the apoptotic potential of propafenone against Candida spp. Conclusion. Taken together, these results indicate that propafenone has the potential for repositioning as an antifungal drug.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals. Hypothesis. Apramycin may have in vitro activity against Mtb. Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value. Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay. Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC 50 of 0.5 μg ml -1 and an MIC 90 of 1 μg ml -1 . We determined the tentative ECOFF value as 1 µg ml -1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides. Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers. Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia. Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing. Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron ( intI1 +aadA6+orfD+qacED1-sul1 ). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU -positive, 9 exoS -positive and 2 exoU/exoS -positive strains. The lasR gene was truncated by IS Ppu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa ; however, the lasR -truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation. Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.

BACKGROUND:Antimicrobial resistance is an emerging global health issue. Data on the epidemiology of multidrug-resistant organisms are scarce for Africa, especially in HIV-infected individuals who often have frequent contact with healthcare. We investigated the prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in stool among HIV-infected children attending an HIV outpatient department in Harare, Zimbabwe. METHODS:We recruited children who were stable on antiretroviral therapy (ART) attending a HIV clinic from August 2014 to June 2015. Information was collected on antibiotic use and hospitalization. Stool was tested for ESBL-E through combination disc diffusion. API20E identification and antimicrobial susceptibility was performed on the positive samples followed by whole genome sequencing. RESULTS:Stool was collected from 175/202 (86.6 %) children. Median age was 11 [inter-quartile range (IQR) 9-12] years. Median time on ART was 4.6 years (IQR 2.4-6.4). ESBL-Es were found in 24/175 samples (13.7 %); 50 % of all ESBL-Es were resistant to amoxicillin-clavulanate, 100 % to co-trimoxazole, 45.8 % to chloramphenicol, 91.6 % to ceftriaxone, 20.8 % to gentamicin and 62.5 % to ciprofloxacin. ESBL-Es variously encoded CTX-M, OXA, TEM and SHV enzymes. The odds of ESBL-E carriage were 8.5 times (95 % CI 2.2-32.3) higher in those on ART for less than one year (versus longer) and 8.5 times (95 % CI 1.1-32.3) higher in those recently hospitalized for a chest infection. CONCLUSION:We found a 13.7 % prevalence of ESBL-E carriage in a population where ESBL-E carriage has not been described previously. Antimicrobial resistance (AMR) in Africa merits further study, particularly given the high HIV prevalence and limited diagnostic and therapeutic options available.

Background. The intensified global challenge of antimicrobial resistance, set against the backdrop of the COVID-19 pandemic, is a cause for major concern. Within healthcare settings, intensive care units are recognized as focal points for Gram-negative infections. The study pursued to assess the prevalence and antimicrobial resistance patterns of critical priority pathogens ( Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacteriaceae, comprising Klebsiella pneumoniae and Escherichia coli ) during both pre- and COVID-19 periods. Gap Statement. The decision to explore this topic stemmed from the urgent need to understand how the exceptional healthcare crisis of COVID-19 affected AMR patterns. Methods. This was an observational retrospective analysis of 1056 clinical specimens obtained from 950 patients who were admitted to the Medical Intensive Care Unit at Kasr Al-Aini Hospital, Cairo University, Egypt. Results. In the period before COVID-19, 342 pathogenic isolates (135 K . pneumoniae , 83 P . aeruginosa , 76 A . baumannii and 48 E. coli ) were obtained from samples collected from 450 patients. Conversely, during the COVID-19 period, 714 isolates (237 K . pneumoniae , 205 A . baumannii , 199 P . aeruginosa and 73 E. coli ) were collected from the same number of patients. In the course of the pandemic, there is a slight increase in A. baumannii and P. aeruginosa infections, whereas E. coli and K. pneumoniae exhibit a distinct trend with a noticeable reduction in infection rates during COVID-19. During the COVID-19 period, a noticeable rise in resistance rates was observed for all antibiotics utilized. The results from Fisher's exact test indicated a substantial increase in resistance towards certain antibiotics. Specifically, a significant rise in resistance was observed for E. coli to ciprofloxacin ( P = 0.00), gentamicin and P. aeruginosa ( P = 0.02), levofloxacin and A. baumannii ( P = 0.01), piperacillin-tazobactam and A. baumannii ( P = 0.04), and piperacillin-tazobactam and P. aeruginosa ( P = 0.01). Conclusion. Our results display how the pandemic impacted bacterial infections and antibiotic resistance, indicating a general increase in resistance rates. These findings are crucial for guiding healthcare practices, emphasizing the need for continued surveillance and potentially checking antibiotic usage schemes.

Introduction. Innovative antifungal therapies are of crucial importance to combat the potentially life-threatening infections linked to the multidrug-resistant fungal pathogen Candida auris . Induction of regulated cell death, apoptosis, could provide an outline for future therapeutics. Human antimicrobial peptides (AMPs), well-known antifungal compounds, have shown the ability to induce apoptosis in pathogenic fungi. Hypothesis/Gap Statement . Although it is known that AMPs possess antifungal activity against C. auris , their ability to induce apoptosis requires further investigations. Aim. This study evaluated the effects of AMPs on the induction of apoptosis in C. auris . Methods. Human neutrophil peptide-1 (HNP-1), human β-Defensins-3 (hBD-3) and human salivary histatin 5 (His 5) were assessed against two clinical C. auris isolates. Apoptosis hallmarks were examined using FITC-Annexin V/PI double labelling assay and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick-end labelling (TUNEL) to detect phosphatidylserine externalization and DNA fragmentation, respectively. Then, several intracellular triggers were studied using JC-10 staining, spectrophotometric assay and 2',7'-dichlorofluorescin diacetate staining to measure the mitochondrial membrane potential, cytochrome-c release and reactive oxygen species (ROS) production, respectively. Results and conclusion. FITC-Annexin V/PI staining and TUNEL analysis revealed that exposure of C. auris cells to HNP-1 and hBD-3 triggered both early and late apoptosis, while His 5 caused significant necrosis. Furthermore, HNP-1 and hBD-3 induced significant mitochondrial membrane depolarization, which resulted in substantial cytochrome c release. In contrast to His 5, which showed minimal mitochondrial depolarization and no cytochrome c release. At last, all peptides significantly increased ROS production, which is related to both types of cell death. Therefore, these peptides represent promising and effective antifungal agents for treating invasive infections caused by multidrug-resistant C. auris.

Introduction. Resistance towards amoxicillin in Helicobacter pylori causes significant therapeutic impasse in healthcare settings worldwide. In Malaysia, the standard H. pylori treatment regimen includes a 14-day course of high-dose proton-pump inhibitor (rabeprazole, 20 mg) with amoxicillin (1000 mg) dual therapy. Hypothesis/Gap Statement. The high eradication rate with amoxicillin-based treatment could be attributed to the primary resistance rates of amoxicillin being relatively low at 0%, however, a low rate of secondary resistance has been documented in Malaysia recently. Aim. This study aims to investigate the amino acid mutations and related genetic variants in PBP1A of H. pylori , correlating with amoxicillin resistance in the Malaysian population. Methodology. The full-length pbp1A gene was amplified via PCR from 50 genomic DNA extracted from gastric biopsy samples of H. pylori -positive treatment-naïve Malaysian patients. The sequences were then compared with reference H. pylori strain ATCC 26695 for mutation and variant detection. A phylogenetic analysis of 50 sequences along with 43 additional sequences from the NCBI database was performed. These additional sequences included both amoxicillin-resistant strains ( n =20) and amoxicillin-sensitive strains ( n =23). Results. There was a total of 21 variants of amino acids, with three of them located in or near the PBP-motif (SKN402-404). The percentages of these three variants are as follows: K403X, 2%; S405I, 2% and E406K, 16%. Based on the genetic markers identified, the resistance rate for amoxicillin in our sample remained at 0%. The phylogenetic examination suggested that H. pylori might exhibit unique conserved pbp1A sequences within the Malaysian context. Conclusions. Overall, the molecular analysis of PBP1A supported the therapeutic superiority of amoxicillin-based regimens. Therefore, it is crucial to continue monitoring the amoxicillin resistance background of H. pylori with a larger sample size to ensure the sustained effectiveness of amoxicillin-based treatments in Malaysia.

Background. Invasive Group B Streptococcus (GBS; Streptococcus agalactiae ) remains a leading cause of infant morbidity and mortality. Intrapartum antibiotic prophylaxis (IAP) has been implemented in many countries with a reduction in early-onset disease, but an effective vaccine may further reduce the disease burden. Candidate vaccines targeting capsular polysaccharides and surface proteins are now in clinical trials. Methods. Using whole-genome sequencing and phenotypic antimicrobial susceptibility testing, we characterized sterile-site GBS isolates recovered from Western Australian infants between 2004 and 2020. Characteristics were compared between three time periods: 2004-2008, 2009-2015 and 2016-2020. Results. A total of 135 isolates were identified. The proportion of serotype III (22.7 % in Period 1 to 47.9 % in Period 3, P =0.04) and clonal complex 17 (13.6-39.6 %, P =0.01) isolates increased over time. Overall coverage of vaccines currently being trialled was >95 %. No isolates were penicillin resistant (MIC>0.25 mg l -1 ), but 21.5 % of isolates had reduced penicillin susceptibility (MIC>0.12 mg l -1 ) and penicillin MIC increased significantly over time ( P =0.04). Clindamycin resistance increased over time to 45.8 % in the latest period. Conclusions. Based on comprehensive characterization of invasive infant GBS in Western Australia, we found that coverage for leading capsular polysaccharide and surface protein vaccine candidates was high. The demonstrated changes in serotype and molecular type highlight the need for ongoing surveillance, particularly with regard to future GBS vaccination programmes. The reduced susceptibility to IAP agents over time should inform changes to antibiotic guidelines.

Introduction. Candida albicans and Staphylococcus aureus are recognized for their development of resistance and biofilm formation. New therapeutic alternatives are necessary in this context. Hypothesis. Etomidate shows potential application in catheters against mixed biofilms of fluconazole-resistant C. albicans and methicillin-resistant S. aureus (MRSA). Aim. The present study aimed to evaluate the activity of etomidate against mixed biofilms of fluconazole-resistant C. albicans and MRSA. Methodology. The action of etomidate against mature biofilms was verified through the evaluation of biomass and cell viability, and its ability to prevent biofilm formation in peripheral venous catheters was determined based on counts of colony forming units (c.f.u.) and confirmed by morphological analysis through scanning electron microscopy (SEM). Results. Etomidate generated a reduction ( P <0.05) in biomass and cell viability starting from a concentration of 250 µg ml -1 . In addition, it showed significant ability to prevent the formation of mixed biofilms in a peripheral venous catheter, as shown by a reduction in c.f.u. SEM revealed that treatment with etomidate caused substantial damage to the fungal cells. Conclusion. The results showed the potential of etomidate against polymicrobial biofilms of fluconazole-resistant C. albicans and MRSA.

Introduction. Salmonella 1,4, [5],12:i:- strains with different antimicrobial resistance profiles have been associated with foodborne disease outbreaks in several countries. In Brazil, S . 1,4, [5],12:i:- was identified as one of the most prevalent serovars in São Paulo State during 2004-2020. Gap Statement. However, few studies have characterized this serovar in Brazil. Aim. This study aimed to determine the antimicrobial resistance profiles of S . 1,4, [5],12:i:- strains isolated from different sources in Southeast Brazil and compare their genetic diversity. Methodology. We analysed 113  S . 1,4, [5],12:i:- strains isolated from humans ( n =99), animals ( n =7), food ( n =5) and the environment ( n =2) between 1983 and 2020. Susceptibility testing against 13 antimicrobials was performed using the disc diffusion method for all the strains. Plasmid resistance genes and mutations in the quinolone resistance-determining regions were identified in phenotypically fluoroquinolone-resistant strains. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for all strains and multilocus sequence typing (MLST) for 40 selected strains. Results. Of the 113 strains, 54.87 % were resistant to at least one antimicrobial. The highest resistance rates were observed against ampicillin (51.33 %), nalidixic acid (39.82 %) and tetracycline (38.05 %). Additionally, 39 (34.51 %) strains were classified as multidrug-resistant (MDR). Nine fluoroquinolone-resistant strains exhibited the gyrA mutation (Ser96→Tyr96) and contained the qnrB gene. The 113 strains were grouped into two clusters using ERIC-PCR, and most of strains were present in one cluster, with a genetic similarity of ≥80 %. Finally, 40 strains were typed as ST19 using MLST. Conclusion. The prevalence of MDR strains is alarming because antimicrobial treatment against these strains may lead to therapeutic failure. Furthermore, the ERIC-PCR and MLST results suggested that most strains belonged to one main cluster. Thus, a prevalent subtype of Salmonella 1,4, [5],12:i:- strains has probably been circulating among different sources in São Paulo, Brazil, over decades.

Introduction. Trichophyton rubrum is a major causative agent of superficial dermatomycoses such as onychomycosis and tinea pedis. Huangqin decoction (HQD), as a classical traditional Chinese medicine formula, was found to inhibit the growth of common clinical dermatophytes such as T. rubrum in our previous drug susceptibility experiments. Hypothesis/Gap Statement. The antifungal activity and potential mechanism of HQD against T. rubrum have not yet been investigated. Aim. The aim of this study was to investigate the antifungal activity and explore the potential mechanism of action of HQD against T. rubrum . Methodology. The present study was performed to evaluate the antifungal activity of HQD against T. rubrum by determination of minimal inhibitory concentrations (MICs), minimal fungicidal concentrations (MFCs), mycelial growth, biomass, spore germination and structural damage, and explore its preliminary anti-dermatophyte mechanisms by sorbitol and ergosterol assay, HPLC-based ergosterol test, enzyme-linked immunosorbent assay and mitochondrial enzyme activity test. Results. HQD was able to inhibit the growth of T. rubrum significantly, with an MIC of 3.125 mg ml -1 and an MFC of 12.5 mg ml -1 . It also significantly inhibited the hyphal growth, conidia germination and biomass growth of T. rubrum in a dose-dependent manner, and induced structural damage in different degrees for T. rubrum cells. HQD showed no effect on cell wall integrity, but was able to damage the cell membrane of T. rubrum by interfering with ergosterol biosynthesis, involving the reduction of squalene epoxidase (SE) and sterol 14α-demethylase P450 (CYP51) activities, and also affect the malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and ATPase activities of mitochondria. Conclusion. These results revealed that HQD had significant anti-dermatophyte activity, which was associated with destroying the cell membrane and affecting the enzyme activities of mitochondria.

Neisseria gonorrhoeae is a bacterial pathogen that causes gonorrhoea, a sexually transmitted infection. Increasing antimicrobial resistance in N. gonorrhoeae is providing motivation to develop new treatment options. In this study, we investigated the effectiveness of the antibiotic ramoplanin as a treatment for N. gonorrhoeae infection. We tested the effectiveness of ramoplanin in vitro against 14 World Health Organization (WHO) reference strains of N. gonorrhoeae and found that it was active against all 14 strains tested. Furthermore, in a Galleria mellonella infection model of N. gonorrhoeae WHO P, we demonstrated that ramoplanin was active in vivo without any evidence of toxicity. This suggests that ramoplanin might be a new promising antibiotic treatment for gonorrhoea.

Introduction. Early detection of carbapenem-resistant Escherichia coli (CREco), categorized as a critical priority pathogen by the World Health Organization (WHO), is crucial in optimizing therapeutic options and to thwart outbreaks in clinical settings. Gap statement. The need of the hour is a diagnostic method that can detect carbapenem resistance conferred by intrinsic or acquired carbapenem resistance mechanisms or both. Aim. The study investigates the performance of a novel screening chromogenic method for detection of CREco. Methodology. Carbapenem-susceptible ( n =23) and non-susceptible ( n =90) E. coli were used to investigate the efficiency of the blue chromogenic test. All of the isolates were received from a tertiary referral hospital in Silchar, India and subjected to the blue chromogenic test and observed for colour change. A colour change from colourless to blue is interpreted as a positive result. The test results were further compared with available methods for detection of carbapenem resistance conferred by carbapenemase production or other carbapenem resistance mechanisms. Results. The blue chromogenic test generated 100 % (CI: 95.98-100 %) sensitive and 100 % (CI: 85.75-100 %) specific results for the detection of CREco with no false-positive or false-negative results. Within 3 h after incubation, the test detects all CREco with carbapenemase activity. Additionally, the blue chromogenic test also positively detected E. coli harbouring carbapenemase variants and with efflux and porin activity, compared to other phenotypic-based approaches. Conclusion. The study highlights a novel method that is highly sensitive and specific, inexpensive, rapid and user-friendly for the detection of CREco. With the surge and expansion of CREco, this sensitive, specific, user-friendly and inexpensive method can be used in laboratories with limited facilities for early detection of CREco, thereby improving infection control along with averting future outbreaks.

Introduction. Helicobacter pylori is the leading cause of peptic ulcers and gastric cancer. The most common treatment regimens use combinations of two or three antibiotics and a proton pump inhibitor (PPI) to suppress stomach acid. The World Health Organization designated clarithromycin-resistant H. pylori as a high priority pathogen for drug development, due to increasing antibiotic resistance globally. Hypothesis/Gap Statement. There is no routine surveillance of H. pylori primary antimicrobial sensitivities in the UK, and published data are lacking. Aim. This study aimed to characterize antimicrobial sensitivities of isolates collected in Nottingham, UK, between 2001 and 2018. Methodology. Gastric biopsy samples were collected, with informed written consent and ethics approval, from 162 patients attending the Queen's Medical Centre in Nottingham for an upper GI tract endoscopy. Antibiotic sensitivity was assessed using E-Tests and a more cost-effective disc diffusion test. Results. The clarithromycin, amoxicillin and levofloxacin disc diffusion tests provided identical results to E-Tests on a subset of 30 isolates. Disparities were observed in the metronidazole test results, however. In total, 241 isolates from 162 patients were tested using at least one method. Of all isolates, 28 % were resistant to clarithromycin, 62 % to metronidazole and 3 % to amoxicillin, which are used in first-line therapies. For those antibiotics used in second- and third-line therapies, 4 % were resistant to levofloxacin and none of the isolates were resistant to tetracycline. Resistance to more than one antibiotic was found in 27 % of isolates. The frequency of patients with a clarithromycin-resistant strain increased dramatically over time: from 16 % between 2001 and 2005 to 40 % between 2011 and 2018 ( P =0.011). For the same time periods, there was also an increase in those with a metronidazole-resistant strain (from 58 to 78 %; P =0.05). The frequencies of clarithromycin and metronidazole resistance were higher in isolates from patients who had previously received eradication therapy, compared to those who had not (40 % versus 77 %, and 80 % versus 92 %, respectively). Of 79 pairs of isolates from the antrum and corpus regions of the same patient's stomach, only six had differences in their antimicrobial susceptibility profiles. Conclusion. Although there was high and increasing resistance to clarithromycin and metronidazole, there was no resistance to tetracycline and the frequencies of amoxicillin and levofloxacin resistance were very low.

Introduction. Ceftobiprole is an advanced-generation broad-spectrum parenteral cephalosporin with activity against MSSA and MRSA. Gap Statement. Ceftobiprole is not currently approved for use to treat S. aureus bacteremia and phase three clinical trials are taking place. Drug approval requires further pre-clinical evidence to support this new indication. Aim. The aim of this study was to evaluate the efficacy of ceftobiprole at the human equivalent efficacious exposure (considering a 500 mg q8h dosing regimen infused over 2 h) against MSSA and MRSA strains in a neutropenic murine model of bacteremia and disseminated infection. Methodology. Two bioluminescent-tagged strains (one MSSA and one MRSA strain) were selected based on their in vitro susceptibility and in vivo growth profiles. Bacterial c.f.u. counts in the blood, lung, kidney, and liver were determined 48 h post-infection or after death. The bioluminescent-tag allowed the visualization of the real-time effects of ceftobiprole therapy compared to the natural progression of the infection in untreated controls. Results. Treatment with ceftobiprole resulted in a significant reduction of the bacterial load with the bioluminescence reduced by 2-log units and bacterial c.f.u. counts reduced by 3- to 6-log units, depending on the organ and bacterial strain. Survival was 100 % in the ceftobiprole-treated group compared to only 0-20 % survival in the untreated control animals for both strains tested. Conclusion. These results suggest that treatment with ceftobiprole using a 500 mg q8h dosing regimen studied in several successful phase three trials, has potential as an antibiotic therapy to treat bacteremia and associated disseminated infections caused by either methicillin-susceptible or methicillin-resistant strains of S. aureus .

Species of the genus Candida, characterized as commensals of the human microbiota, are opportunistic pathogens capable of generating various types of infections with high associated costs. Considering the limited pharmacological arsenal and the emergence of antifungal-resistant strains, the repositioning of drugs is a strategy used to search for new therapeutic alternatives, in which minocycline and doxycycline have been evaluated as potential candidates. Thus, the objective was to evaluate the in vitro antifungal activity of two tetracyclines, minocycline and doxycycline, and their possible mechanism of action against fluconazole-resistant strains of Candida spp. The sensitivity test for antimicrobials was performed using the broth microdilution technique, and the pharmacological interaction with fluconazole was also analysed using the checkerboard method. To analyse the possible mechanisms of action, flow cytometry assays were performed. The minimum inhibitory concentration obtained was 4-427 µg ml -1 for minocycline and 128-512 µg ml -1 for doxycycline, and mostly indifferent and additive interactions with fluconazole were observed. These tetracyclines were found to promote cellular alterations that generated death by apoptosis, with concentration-dependent reactive oxygen species production and reduced cell viability. Therefore, minocycline and doxycycline present themselves as promising study molecules against Candida spp.

Introduction. Tigecycline is one of the important antibiotics available for treating infection caused by multiple-drug resistant pathogens. However, the conventional AST methods which are commonly used in clinical microbiology laboratories usually lead to false intermediate or resistant results in testing tigecycline susceptibility, and further mislead clinical antimicrobial therapies. Hypothesis. The modified Kirby-Bauer disc diffusion (mKB) method was performed based on the traditional standard Kirby-Bauer disc diffusion (sKB) method. Aim . To evaluate a modified Kirby-Bauer disc diffusion (mKB) method for tigecycline susceptibility testing, for the purpose of providing accurate tigecycline susceptibility results in clinical practice. Methodology. A total of 4271 nonduplicate clinical strains were isolated from 37 hospitals across China to perform the mKB method, standard Kirby-Bauer disc diffusion (sKB) method, comparing with the reference broth microdilution (BMD) according to the CLSI. Parameters of categorical agreement (CA), minor errors (mE), major errors (ME), and very major errors (VME) were used in this methodological evaluation research. Results. BMD testing showed that 91.3-98.9 % of the A. baumannii , K. pneumoniae , E. coli , E. cloacae , S. marcescens , and C. freundii strains were susceptible, while 0-3.1% strains were resistant to tigecycline. When testing A. baumannii , mKB demonstrated higher CA than sKB (90.6 % vs 44.8 %) compared to reference BMD. The mE (9.0 % vs 45.2 %), ME (0.5 % vs 10.6 %) and VME (both 0 %) all satisfied the acceptability criteria. mKB also showed higher CA (87.2 % vs 52.0 %) than sKB in comparison with BMD when testing Enterobacterales (mainly K. pneumonia ). The ME (0.45 % vs 8.1 %) and VME (both 0 %) but not mE (12.4 % vs 40.4 %) met the acceptability criteria. Conclusion. The mKB method can test bacterial susceptibility to tigecycline more accurately than sKB. For the tigecycline-intermediate or -resistant strains by sKB method, BMD or mKB method should be used to verify the results and report reliable tigecycline susceptibility results.

Introduction. Linezolid is an effective therapeutic option for treating severe infections caused by multidrug-resistant Gram-positive organisms. Several mechanisms have been reported to be responsible for resistance to this antibiotic. Hypothesis or Gap Statement. Although several mechanisms of linezolid resistance have been reported in Staphylococcus haemolyticus , the prevalence and potential for horizontal transfer of resistance genes have not been fully characterized, particularly among S. haemolyticus isolates from India. Aim. To perform whole-genome sequencing (WGS) of linezolid-resistant S. haemolyticus isolates to characterize the resistance mechanisms. Methodology. WGS was performed for 16 linezolid-resistant S. haemolyticus isolates to check for the presence of cfr , optrA and poxtA genes and mutations in 23S rRNA and ribosomal proteins (L3, L4 and L22) that are possible mechanisms implicated in linezolid resistance. Sequence types were identified using MLST finder. The minimum inhibitory concentration (MIC) of linezolid was determined using the E-test method. Polymerase chain reaction (PCR) was carried out for the detection of the cfr gene. Results. The study documented three different mechanisms of linezolid resistance in S. haemolyticus . Thirteen of the 16 isolates were phenotypically resistant to linezolid, of which 12 were positive for the cfr gene. The G2603T mutation in 23S rRNA was found in the majority of the isolates ( n =13). Ten isolates had the R138V mutation in L3 ribosomal protein. Twelve isolates with the cfr gene in combination with either G2603T or R138V mutations displayed extremely high MIC values. Surprisingly, three phenotypically sensitive isolates were found to be positive for the cfr gene but negative for other resistance mechanisms. Importantly, in almost half of the isolates the cfr gene was present on a plasmid. ST3 and ST1 were found to be the predominant sequence types. Conclusion. All phenotypically resistant isolates exhibited two or three linezolid resistance mechanisms. The cfr gene was found on plasmids in many isolates, demonstrating its potential for horizontal transfer to more pathogenic organisms.