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Malaria elimination requires interventions able to target both the asexual blood stage (ABS) parasites and transmissible gametocyte stages of Plasmodium falciparum. Lead antimalarial candidates are evaluated against clinical isolates to address key concerns regarding efficacy and to confirm that the current, circulating parasites from endemic regions lack resistance against these candidates. While this has largely been performed on ABS parasites, limited data are available on the transmission-blocking efficacy of compounds with multistage activity. Here, we evaluated the efficacy of lead antimalarial candidates against both ABS parasites and late-stage gametocytes side-by-side, against clinical P. falciparum isolates from southern Africa. We additionally correlated drug efficacy to the genetic diversity of the clinical isolates as determined with a panel of well-characterized, genome-spanning microsatellite markers. Our data indicate varying sensitivities of the isolates to key antimalarial candidates, both for ABS parasites and gametocyte stages. While ABS parasites were efficiently killed, irrespective of genetic complexity, antimalarial candidates lost some gametocytocidal efficacy when the gametocytes originated from genetically complex, multiple-clone infections. This suggests a fitness benefit to multiclone isolates to sustain transmission and reduce drug susceptibility. In conclusion, this is the first study to investigate the efficacy of antimalarial candidates on both ABS parasites and gametocytes from P. falciparum clinical isolates where the influence of parasite genetic complexity is highlighted, ultimately aiding the malaria elimination agenda.

Staphylococcus aureus poses a significant threat as an opportunistic pathogen in humans, and animal medicine, particularly in the context of hospital-acquired infections (HAIs). Effective treatment is a significant challenge, contributing substantially to the global health burden. While antibiotic therapy remains the primary approach for staphylococcal infections, its efficacy is often compromised by the emergence of resistant strains and biofilm formation. The anticipated solution is the discovery and development of new antibacterial agents. However, this is a time consuming and expensive process with limited success rates. One potential alternative for addressing this challenge is the repurposing of existing antibiotics. This study investigated the potential of rifabutin (RFB) as a repurposed antibiotic for treating S. aureus infections. The minimum inhibitory concentration (MIC) of rifabutin was assessed by the broth microdilution method, in parallel to vancomycin, against 114 clinical isolates in planktonic form. The minimum biofilm inhibitory concentration (MBIC50) was determined by an adaptation of the broth microdilution method, followed by MTT assay, against a subset of selected 40 clinical isolates organized in biofilms. The study demonstrated that RFB MIC ranged from 0.002 to 6.250 µg/mL with a MIC50 of 0.013 µg/mL. RFB also demonstrated high anti-biofilm activity in the subset of 40 clinical isolates, with confirmed biofilm formation, with no significant MBIC50 differences observed between the MSSA and MRSA strains, in contrast to that observed for the VAN. These results highlight the promising efficacy of RFB against staphylococcal clinical isolates with different resistance patterns, whether in planktonic and biofilm forms. [ABSTRACT FROM AUTHOR]

Onychomycosis is a common fungal infection that affects the nails and accounts for approximately 50% of all nail diseases. The main pathogens involved include dermatophytes, such as Trichophyton rubrum, members of the T. mentagrophytes complex, and emerging pathogens in this infection, T. schoenleinii and T. tonsurans. Tea tree (Melaleuca alternifolia Cheel) essential oil (EO) has been proposed as a promising natural alternative to traditional treatments due to its antimicrobial properties. Among its more than 100 compounds, terpinen-4-ol is one of the main contributors to the antifungal action of this EO. To determine the antifungal activity of tea tree EO against dermatophytes, we designed an in vitro study using EUCAST-AFST protocols to obtain the values of MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) of several commercial M. alternifolia Cheel EOs against three species of dermatophytes isolated from clinical samples with suspected toenail onychomycosis. The results showed that the microorganism most sensitive to the action of the EO was T. rubrum, which had an MIC value more than 13 times lower than the value obtained for T. schoenleinii (0.4% v/v), the most resistant isolate. No differences in antifungal activity were observed by the analysed EOs or between the MIC and MFC values. These in vitro results suggest that tea tree EO is a viable option for the alternative treatment of onychomycosis, although clinical studies are needed to confirm the long-term antifungal activity, safety and efficacy of the oils studied in a clinical context. [ABSTRACT FROM AUTHOR]

Background: In addition to antibiotic resistance, persistence is another cause of treatment failure in bacterial infections, representing a significant public health concern. Due to a lack of adequate data on clinical isolates, this study was initiated to investigate persistence in clinical isolates in Burkina Faso. Methods: Eighty (80) clinical isolates, including 32 Pseudomonas aeruginosa, 41 Staphylococcus aureus, and 7 Salmonella sp. obtained from clinical laboratories in Burkina Faso, were analyzed to assess their susceptibility to ciprofloxacin and gentamicin, as well as to determine the presence of persistence genes. The effects of ciprofloxacin and gentamicin on persister formation were evaluated by conducting colony counts at 1, 3, 5, 7, and 20 h after exposing the bacteria to high concentrations of these antibiotics. Results: Results showed high sensitivity to both antibiotics (72.5% for ciprofloxacin and 82.5% for gentamicin). Persister formation occurred in Staphylococcus aureus with gentamicin and in Salmonella sp. with ciprofloxacin, while Pseudomonas aeruginosa did not form persisters. The mazF gene was found in 28.13% of P. aeruginosa and 2.44% of S. aureus isolates, and the hipA gene in 28.57% of Salmonella sp. None of the relE1 or relE2 genes were detected. Conclusions: The study revealed high sensitivity in clinical bacterial isolates to ciprofloxacin and gentamicin. Staphylococcus aureus and Salmonella sp. showed persister formation under antibiotic stress, with low frequencies of the studied persistence genes. These findings enhance understanding of clinical bacterial behavior and inform strategies against antibiotic-resistant infections. [ABSTRACT FROM AUTHOR]

Background: Nosocomial infections are a global problem in hospitals all around the world. It is considered a major health problem, especially in developing countries. The increase in the patient's stay in hospitals has increased the mortality rate, and consequently, the costs drastically increase. The main purpose of using disinfectants in the hospital environment is to reduce the risk of nosocomial infections. Ethylene diamine tetra acetic acid (EDTA) causes lysis and increases susceptibility to antimicrobial agents in the planktonic form of bacteria. This substance affects the permeability of the outer membrane of bacteria. It also prevents the formation of biofilms by bacteria. Materials and methods: In the current study, 120 isolates of Acinetobacter baumannii (A. baumannii) were confirmed by phenotypic and genotypic methods. Antibiogram was performed and then the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of isolates against 5% sodium hypochlorite, ethanol %70, sayasept-HP 2%, chlorhexidine 2%, dettol 4/8% were evaluated. In addition, the disinfectant effect was re-evaluated with the mixture of EDTA solution. All isolates were examined for biofilm presence by crystal violet staining method in triplicates and repeated three times for each strain. Also for all isolates detection of efflux pump genes (Qac-E, qacE-Δ1, SUG-E) by PCR technique was done. Results: Antibiogram results of A. baumannii showed that 6.7% were Multi-drug-resistant (MDR), and 89.2% were Extensively drug-resistant (XDR) isolates. The highest effect of disinfectants was related to 5% sodium hypochlorite, and the least effect was 70% ethanol. EDTA increases the efficacy of selected disinfectants significantly. The highest prevalence of the efflux pump genes was related to SUG-E (95%) and Qac-E (91.7%), and, the qacE-Δ1 gene with 12.5%. The biofilm production rate was 91.3% among all isolates. Conclusion: The best and safest way to disinfect hospital floors and surfaces is to choose the right disinfectants, and learn how to use them properly. In this study, a mixture of disinfectants and EDTA had a significant effect on bactericidal activity. it was found that improper use of disinfectants, especially the use of sub-inhibitory dilutions, increases the resistance of bacteria to disinfectants. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas aeruginosa is a key pathogen in cystic fibrosis (CF) associated infections, known for its biofilm-forming ability and resistance to multiple antibiotics. Understanding the relationship between biofilm production and antibiotic resistance profiles in clinical isolates can guide treatment strategies. Aims and Objectives: The aims of this study were to investigate the biofilm-forming abilities and antibiotic resistance profiles of clinical P. aeruginosa isolates from patients with CF and to determine the relationship between these two factors and their impact on treatment efficacy. Materials and Methods: A cross-sectional study was conducted on 100 clinical isolates of P. aeruginosa from CF patients. Biofilmforming capacity was categorized as strong, moderate, or weak based on quantitative assays. Antibiotic resistance was assessed for ciprofloxacin, tobramycin, ceftazidime, meropenem, and piperacillin/tazobactam. Multi-drug resistance was defined as resistance to three or more antibiotic classes. Statistical correlations between biofilm-forming capacity and resistance levels were evaluated using the Chi-square tests. Results: Fifty-six percentages of isolates were strong biofilm producers, while 34% and 10% were moderate and weak producers, respectively. The highest antibiotic resistance was observed against ciprofloxacin (60%), followed by tobramycin (50%) and ceftazidime (45%). Forty percentages of the isolates were classified as multi-drug resistant. Strong biofilm producers demonstrated a significantly high correlation with antibiotic resistance (P<0.05). Two predominant clonal groups were identified among the isolates, suggesting a possible clonal spread of resistance traits. Conclusion: The study confirms a strong association between robust biofilm production and heightened antibiotic resistance in P. aeruginosa isolates from CF patients. These findings highlight the need for targeted therapeutic strategies to disrupt biofilm formation and curb resistance spread. [ABSTRACT FROM AUTHOR]

Objectives In this study, we performed comparative genomic and transcriptomic analysis of clinical isolates of Mycobacterium tuberculosis collected from patients with drug-susceptible tuberculosis (DS-TB). The clinical isolates were categorized based on treatment duration: standard 6 months or >6 months. Methods Study participants were recruited from a 2016 to 2018 tuberculosis cohort, and clinical M. tuberculosis isolates were collected from the sputum of patients with tuberculosis. We analyzed the genome and transcriptome of the isolated M. tuberculosis. Results Genomic analysis revealed a specific non-synonymous single-nucleotide polymorphism in pe_pgrs9 and ppe34, exclusive to the group treated for >6 months. Transcriptomic analysis revealed increased expression of various virulence-associated protein family genes and decreased expression of ribosomal protein genes and ppe38 genes in the group treated for >6 months. Conclusion The identified genetic variation and gene expression patterns may influence treatment outcomes by modulating host immune responses, increasing virulence, and potentially contributing to persister cell formation in M. tuberculosis. This study provides insights into the genetic and transcriptomic factors associated with prolonged DS-TB treatment. However, our study identified molecular characteristics using a small sample size, and further detailed studies are warranted.

Abstract Background In addition to antibiotic resistance, persistence is another cause of treatment failure in bacterial infections, representing a significant public health concern. Due to a lack of adequate data on clinical isolates, this study was initiated to investigate persistence in clinical isolates in Burkina Faso. Methods Eighty (80) clinical isolates, including 32 Pseudomonas aeruginosa, 41 Staphylococcus aureus, and 7 Salmonella sp. obtained from clinical laboratories in Burkina Faso, were analyzed to assess their susceptibility to ciprofloxacin and gentamicin, as well as to determine the presence of persistence genes. The effects of ciprofloxacin and gentamicin on persister formation were evaluated by conducting colony counts at 1, 3, 5, 7, and 20 h after exposing the bacteria to high concentrations of these antibiotics. Results Results showed high sensitivity to both antibiotics (72.5% for ciprofloxacin and 82.5% for gentamicin). Persister formation occurred in Staphylococcus aureus with gentamicin and in Salmonella sp. with ciprofloxacin, while Pseudomonas aeruginosa did not form persisters. The mazF gene was found in 28.13% of P. aeruginosa and 2.44% of S. aureus isolates, and the hipA gene in 28.57% of Salmonella sp. None of the relE1 or relE2 genes were detected. Conclusions The study revealed high sensitivity in clinical bacterial isolates to ciprofloxacin and gentamicin. Staphylococcus aureus and Salmonella sp. showed persister formation under antibiotic stress, with low frequencies of the studied persistence genes. These findings enhance understanding of clinical bacterial behavior and inform strategies against antibiotic-resistant infections.

Abstract Background Nosocomial infections are a global problem in hospitals all around the world. It is considered a major health problem, especially in developing countries. The increase in the patient’s stay in hospitals has increased the mortality rate, and consequently, the costs drastically increase. The main purpose of using disinfectants in the hospital environment is to reduce the risk of nosocomial infections. Ethylene diamine tetra acetic acid (EDTA) causes lysis and increases susceptibility to antimicrobial agents in the planktonic form of bacteria. This substance affects the permeability of the outer membrane of bacteria. It also prevents the formation of biofilms by bacteria. Materials and methods In the current study, 120 isolates of Acinetobacter baumannii (A. baumannii) were confirmed by phenotypic and genotypic methods. Antibiogram was performed and then the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of isolates against 5% sodium hypochlorite, ethanol %70, sayasept-HP 2%, chlorhexidine 2%, dettol 4/8% were evaluated. In addition, the disinfectant effect was re-evaluated with the mixture of EDTA solution. All isolates were examined for biofilm presence by crystal violet staining method in triplicates and repeated three times for each strain. Also for all isolates detection of efflux pump genes (Qac-E, qacE-Δ1, SUG-E) by PCR technique was done. Results Antibiogram results of A. baumannii showed that 6.7% were Multi-drug-resistant (MDR), and 89.2% were Extensively drug-resistant (XDR) isolates. The highest effect of disinfectants was related to 5% sodium hypochlorite, and the least effect was 70% ethanol. EDTA increases the efficacy of selected disinfectants significantly. The highest prevalence of the efflux pump genes was related to SUG-E (95%) and Qac-E (91.7%), and, the qacE-Δ1 gene with 12.5%. The biofilm production rate was 91.3% among all isolates. Conclusion The best and safest way to disinfect hospital floors and surfaces is to choose the right disinfectants, and learn how to use them properly. In this study, a mixture of disinfectants and EDTA had a significant effect on bactericidal activity. it was found that improper use of disinfectants, especially the use of sub-inhibitory dilutions, increases the resistance of bacteria to disinfectants.

Background: Pseudomonas aeruginosa is a key pathogen in cystic fibrosis (CF) associated infections, known for its biofilm-forming ability and resistance to multiple antibiotics. Understanding the relationship between biofilm production and antibiotic resistance profiles in clinical isolates can guide treatment strategies. Aims and Objectives: The aims of this study were to investigate the biofilm-forming abilities and antibiotic resistance profiles of clinical P. aeruginosa isolates from patients with CF and to determine the relationship between these two factors and their impact on treatment efficacy. Materials and Methods: A cross-sectional study was conducted on 100 clinical isolates of P. aeruginosa from CF patients. Biofilm-forming capacity was categorized as strong, moderate, or weak based on quantitative assays. Antibiotic resistance was assessed for ciprofloxacin, tobramycin, ceftazidime, meropenem, and piperacillin/tazobactam. Multi-drug resistance was defined as resistance to three or more antibiotic classes. Statistical correlations between biofilm-forming capacity and resistance levels were evaluated using the Chi-square tests. Results: Fifty-six percentages of isolates were strong biofilm producers, while 34% and 10% were moderate and weak producers, respectively. The highest antibiotic resistance was observed against ciprofloxacin (60%), followed by tobramycin (50%) and ceftazidime (45%). Forty percentages of the isolates were classified as multi-drug resistant. Strong biofilm producers demonstrated a significantly high correlation with antibiotic resistance (P

Background & Aims: Citrobacter, a member of the Enterobacteriaceae family known to be a normal intestinal flora and an opportunistic pathogen is now increasingly found to cause a variety of infections in community as well as hospital settings. It was initially considered a low virulence pathogen but is now found to cause multi-drug resistant infections with high morbidity and mortality. To determine the prevalence of infections caused by Citrobacter spp. and theirAntibiotic sensitivity pattern. Material & Methods: A Laboratory records-based Cross-sectional study was undertaken retrospectively wherein the laboratory data about 50 Citrobacter spp. isolates obtained from 1628 clinical samples processed over 18 months were retrieved and analyzed at Department of Microbiology, Government Medical College-Datia, Central India in January 2024. Result: Out of the 1628 samples processed, significant bacterial growth was reported in 770 samples out of which 50 were found to be positive for Citrobacter spp. (6.4%). Isolation rate for Citrobacter spp. was reported to be 3.1 %.The majority of isolates were obtained from Urine (49%) and pus (45%) samples with C. koseri being dominant in urine and C. freundii in pus. Amongst these isolates, 52% were C.koseri, 46% were C.freundii and 4% were other species. Conclusion: The findings of the study conducted in January 2024 at Datia (Madhya Pradesh) indicate a change in susceptibility trends of emerging pathogens like Citrobacter spp. exhibiting resistance to routinely prescribed antimicrobials, stressing the need for an appropriate action plan involving periodic surveillance, antimicrobial stewardship, and strict implementation of infection control practices.

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings. [ABSTRACT FROM AUTHOR]

Although cases of Legionnaires' disease are notifiable, data on the phenotypic and genotypic characterisation of clinical isolates are limited. This retrospective study aims to report the results of the characterisation of Legionella clinical isolates in Spain from 2012 to 2022. Monoclonal antibodies from the Dresden panel were used for phenotypic identification of Legionella pneumophila. Genotypic characterisation and sequence type assignment were performed using the Sequence-Based Typing scheme. Of the 1184 samples, 569 were identified as Legionella by culture. Of these, 561 were identified as L. pneumophila, of which 521 were serogroup 1. The most common subgroups were Philadelphia (n = 107) and Knoxville (n = 106). The SBT analysis revealed 130 different STs, with the most common genotypes being ST1 (n = 87), ST23 (n = 57), ST20 (n = 30), and ST42 (n = 29). Knoxville has the highest variability with 32 different STs. ST23 is mainly found in Allentown/France (n = 46) and ST42 in Benidorm (n = 18), whereas ST1 is widely distributed. The results demonstrate that clinical isolates show high genetic diversity, although only a few sequence types (STs) are responsible for most cases. However, outbreaks can also occur with rare genotypes. More data on LD and associated epidemiological studies are needed to establish the risk of an isolate causing outbreak in the future. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus poses a significant threat as an opportunistic pathogen in humans, and animal medicine, particularly in the context of hospital-acquired infections (HAIs). Effective treatment is a significant challenge, contributing substantially to the global health burden. While antibiotic therapy remains the primary approach for staphylococcal infections, its efficacy is often compromised by the emergence of resistant strains and biofilm formation. The anticipated solution is the discovery and development of new antibacterial agents. However, this is a time consuming and expensive process with limited success rates. One potential alternative for addressing this challenge is the repurposing of existing antibiotics. This study investigated the potential of rifabutin (RFB) as a repurposed antibiotic for treating S. aureus infections. The minimum inhibitory concentration (MIC) of rifabutin was assessed by the broth microdilution method, in parallel to vancomycin, against 114 clinical isolates in planktonic form. The minimum biofilm inhibitory concentration (MBIC50) was determined by an adaptation of the broth microdilution method, followed by MTT assay, against a subset of selected 40 clinical isolates organized in biofilms. The study demonstrated that RFB MIC ranged from 0.002 to 6.250 μg/mL with a MIC50 of 0.013 μg/mL. RFB also demonstrated high anti-biofilm activity in the subset of 40 clinical isolates, with confirmed biofilm formation, with no significant MBIC50 differences observed between the MSSA and MRSA strains, in contrast to that observed for the VAN. These results highlight the promising efficacy of RFB against staphylococcal clinical isolates with different resistance patterns, whether in planktonic and biofilm forms.

Purpose: Antibiotics play an important role in treating periodontal diseases. Due to the effectiveness of antibiotic therapies, their usage in dentistry has significantly increased. The aim of this study focused on the in-vitro susceptibility of different gram-negative oral bacteria species -- which are associated with periodontal diseases (Fusobacterium spp., Capnocytophaga spp. and Leptotrichia buccalis) and have different geographical origins (Asia and Europe) -- against antimicrobials that are clinically relevant in dental therapy. Materials and Methods: A total of 45 strains were tested (29 Fusobacterium spp., 13 Capnocytophaga spp. and 3 L. buccalis) that were either isolated from Chinese patients or were obtained from different strain collections. Their antimicrobial susceptibility to the antimicrobial agents benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, clindamycin, doxycycline, tetracycline and metronidazole was tested using the E-Test. Strains with particular resistance to penicillin, clindamycin and metronidazole were further analysed for resistance genes. Results: All tested bacterial isolates were sensitive to amoxicillin, amoxicillin-clavulanic acid, doxycycline and tetracycline, but showed variable sensitivity towards other antibiotics such as benzylpenicillin, ciprofloxacin, moxifloxacin, clindamycin and metronidazole. Conclusion: The results of the present study suggest that certain periodontal disease-related bacterial strains can be resistant towards antimicrobial agents commonly used in adjuvant periodontal therapy. [ABSTRACT FROM AUTHOR]

Introduction: Mycoplasma hominis (M. hominis) belongs to the class Mollicutes, characterized by a very small genome size, reduction of metabolic pathways, including transcription factors, and the absence of a cell wall. Despite this, they adapt well not only to specific niches within the host organism but can also spread throughout the body, colonizing various organs and tissues. The adaptation mechanisms of M. hominis, as well as their regulatory pathways, are poorly understood. It is known that, when adapting to adverse conditions, Mycoplasmas can undergo phenotypic switches that may persist for several generations. Methods: To investigate the adaptive properties of M. hominis related to survival in the host, we conducted a comparative phenotypic and proteogenomic analysis of eight clinical isolates of M. hominis obtained from patients with urogenital infections and the laboratory strain H-34. Results: We have shown that clinical isolates differ in phenotypic features from the laboratory strain, form biofilms more effectively and show resistance to ofloxacin. The comparative proteogenomic analysis revealed that, unlike the laboratory strain, the clinical isolates possess several features related to stress survival: they switch carbon metabolism, activating the energetically least advantageous pathway of nucleoside utilization, which allows slowing down cellular processes and transitioning to a starvation state; they reconfigure the repertoire of membrane proteins; they have integrative conjugative elements in their genomes, which are key mediators of horizontal gene transfer. The upregulation of the methylating subunit of the restriction-modification (RM) system type I and the additional components of RM systems found in clinical isolates suggest that DNA methylation may play a role in regulating the adaptation mechanisms of M. hominis in the host organism. It has been shown that based on the proteogenomic profile, namely the genome sequence, protein content, composition of the RM systems and additional subunits HsdM, HsdS and HsdR, composition and number of transposable elements, as well as the sequence of the main variable antigen Vaa, we can divide clinical isolates into two phenotypes: typical colonies (TC), which have a high growth rate, and atypical (aTC) mini-colonies, which have a slow growth rate and exhibit properties similar to persisters. Discussion: We believe that the key mechanism of adaptation of M. hominis in the host is phenotypic restructuring, leading to a slowing down cellular processes and the formation of small atypical colonies. This is due to a switch in carbon metabolism and activation the pathway of nucleoside utilization. We hypothesize that DNA methylation may play a role in regulating this switch. [ABSTRACT FROM AUTHOR]

The heterogenicity of antimicrobial resistance genes described in clinically significant bacterial isolates and their potential role in reducing the efficacy of classically effective antibiotics pose a major challenge for global healthcare, especially in infections caused by Gram-negative bacteria. We analyzed 112 multidrug-resistant (MDR) isolates from clinical samples in order to detect high resistance profiles, both phenotypically and genotypically, among four Gram-negative genera (Acinetobacter, Escherichia, Klebsiella, and Pseudomonas). We found that 9.8% of the total selected isolates were classified as extensively drug-resistant (XDR) (six isolates identified as A. baumannii and five among P. pneumoniae isolates). All other isolates were classified as MDR. Almost 100% of the isolates showed positive results for blaOXA-23 and blaNDM-1 genes among the A. baumannii samples, one resistance gene (blaCTX-M) among E. coli, and two genetic determinants (blaCTX-M and aac(6′)-Ib) among Klebsiella. In contrast, P. aeruginosa showed just one high-frequency antibiotic resistance gene (dfrA), which was present in 68.42% of the isolates studied. We also describe positive associations between ampicillin and cefotaxime resistance in A. baumannii and the presence of blaVEB and blaGES genes, as well as between the aztreonam resistance phenotype and the presence of blaGES gene in E. coli. These data may be useful in achieving a better control of infection strategies and antibiotic management in clinical scenarios where these multidrug-resistant Gram-negative pathogens cause higher morbidity and mortality. [ABSTRACT FROM AUTHOR]

Medicinal plants are common in our environment and have been useful in traditional medicine. The present study was conducted to evaluate the mineral content, the phytochemical, and antimicrobial properties of Carica papaya seed extracts on clinical isolates. The C. papaya seeds were sourced from fruits sellers from the Ekosodin community in Ovia North East Local Government, Benin City, Edo State. The seeds were air-dried for 14 days. The extract was obtained by maceration using distilled water and ethanol as solvents. The results of the mineral composition for both aqueous and ethanolic extracts indicated that sodium, potassium, calcium, zinc, copper, phosphorus, nitrogen, chromium, and iron were present. The phytochemical screening for both aqueous and ethanolic extracts indicated the presence of compounds such as saponins, phenolics, alkaloids, and flavonoids. The antimicrobial assay revealed that C. papaya seed extracts had good antimicrobial properties with a minimum inhibition of 100 mg/mL observed in Staphylococcus epidermidis and Bacillus subtilis for the aqueous extract, 1.04 mg/ml S. epidermidis and B. subtilis , and 2.08 mg/ml Candida spp. for the ethanol extract. The minimum bactericidal concentration of the ethanol extract in this study was 4.16 mg/ml for S. epidermidis and 8.33 mg/ml for B. subtilis , and the minimum fungicidal concentration was 8.33 mg/ml for Candida spp. The results from this study indicated that C. papaya seeds possess sufficient mineral components and phytochemical components, indicating their potential use as supplementary antimicrobial agents and essential nutrients for both humans and animals. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 μg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 μg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Acinetobacter spp. is associated with nosocomial infections in intensive care unit patients, resulting in high mortality. Although Acinetobacter spp. represent a serious public health problem worldwide, there are a few studies related to the presence of carbapenemases in health care facilities and other environmental settings in Ecuador. The main aim of this study was to characterize the carbapenem-resistant Acinetobacter spp. isolates obtained from four hospitals (52) and from five rivers (27) close to Quito. We used the disc diffusion and EDTA sinergy tests to determine the antimicrobial susceptibility and the production of metallo β-lactamases, respectively. We carried out a multiplex PCR of gyrB gene and the sequencing of partial rpoB gene to bacterial species identification. We performed molecular screening of nine carbapenem-resistant genes (blaSPM, blaSIM, blaGIM, blaGES, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, and blaOXA-143) by multiplex PCR, followed by identification using sequencing of blaOXA genes. Our findings showed that carbapenem-resistant A. baumannii were the main species found in health care facilities and rivers. Most of the clinical isolates came from respiratory tract samples and harbored blaOXA-23, blaOXA-366, blaOXA-72, blaOXA-65, blaOXA-70, and blaOXA-143- like genes. The river isolates harbored only the blaOXA-51 and probably blaOXA-259 genes. We concluded that the most predominant type of carbapenem genes among isolates were both blaOXA-23 and blaOXA-65 among A. baumannii clinical isolates. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to investigate plasmid profiles and antibiotic resistance patterns of multidrug resistant Pseudomonas aeruginosa strains isolated from different clinical specimens in Jordan. Methodology and results: Pseudomonas aeruginosa strains were isolated from different clinical specimens from different hospitals and primary health care centers. The antimicrobial susceptibility of P. aeruginosa isolates was determined using the disc diffusion method against 16 commonly used antimicrobial drugs. Plasmid DNAs were extracted from lysed P. aeruginosa cells using the plasmid alkaline lysis method and visualized using agarose gel electrophoresis followed by ethidium bromide staining. The isolated strains of P. aeruginosa were resistant to cefepime (90%), meropenem (70%), ceftazidime (60%), piperacillin (55%), aztreonam (50%), ciprofloxacin and tobramycin (35%), gentamicin (29%), imipenem and amikacin (20%). All the isolates were sensitive to colistin. Plasmid analysis of the clinical isolates showed the presence of 0 to 3 plasmids with a size range of 1 to 25 kb compared to the standard strain of Escherichia coli (ATCC 25922). Conclusion, significance and impact of study: The results obtained in this study showed some correlation between the patterns of antibiotic resistance and plasmid profiles. [ABSTRACT FROM AUTHOR]

Introduction: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. are microorganisms referred as the ESKAPE group pathogens. These microorganisms have generated great concern in health institutions around the world since most of them have resistance to multiple antibiotics and cause most infections associated with healthcare, as well as community infections. The aim of this study was the analysis of antibiotic resistance in microorganisms of the ESKAPE group, recovered from clinical samples in 11 health institutions from Hermosillo and Ciudad Obregón in the State of Sonora, México, during the period from 2019 to 2020. Methods: A cross-sectional, descriptive, observational, and temporality epidemiological study was carried out. A comparative and statistical analysis of antibiotic resistance was carried out using the chi-square test, and small values were analyzed using Fisher's exact test p ≤ 0.05. Results and discussion: All the ESKAPE group microorganisms showed significant differences in antibiotic resistance percentages between both cities. High resistance percentages for some antibiotics, like cephalosporins and ciprofloxacin were detected for Klebsiella pneumoniae and Acinetobacter baumannii. [ABSTRACT FROM AUTHOR]

The opportunistic fungus Candida albicans is the leading cause of invasive candidiasis in immune-compromised individuals. Drugs from the echinocandin (ECN) class, including caspofungin, are used as a first line of therapy against invasive candidiasis. The only known mechanism of clinical resistance to ECNs is point mutations in the FKS1 gene, which encodes the drug target. However, many clinical isolates developed decreased ECN susceptibilities in the absence of resistance-associated FKS1 mutations. We have identified 15 C. albicans genes that contribute to decreased drug susceptibility. We explored the expression of these 15 genes in clinical isolates with different levels of ECN susceptibility. We found that these 15 genes are expressed in clinical isolates with or without FKS1 mutations, including those strains that are less susceptible to ECNs. In addition, FKS1 expression was increased in such less susceptible isolates compared to highly susceptible isolates. Similarities of gene expression patterns between isolates with decreased ECN susceptibilities in the absence of FKS1 mutations and clinically resistant isolates with mutations in FKS1 suggest that clinical isolates with decreased ECN susceptibilities may be a precursor to development of resistance. [ABSTRACT FROM AUTHOR]

The genus Acanthamoeba comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on Acanthamoeba sequence types and alleles are available in Italy. In the present study, we analyzed all Acanthamoeba sequences deposited from Italy with new positive Acanthamoeba clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 Acanthamoeba sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The Acanthamoeba T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of Acanthamoeba isolates throughout Italy, reaffirming its well-recognized ubiquity. Acanthamoeba isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales. [ABSTRACT FROM AUTHOR]

ObjectivesTo evaluate the in vitro antimicrobial susceptibilities of Gram-positive and Gram-negative isolates from patients in Jordan between 2010 and 2021, through the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme.MethodsMedical centres in Jordan collected bacterial isolates from hospitalised patients with defined infection sources between 2010 and 2021 (no isolates collected in 2014). Antimicrobial susceptibility was interpreted using CLSI standards. FDA-approved breakpoints were applied for tigecycline. The identification of β-lactamase genes was performed for a proportion of isolates using multiplex PCR assays.ResultsMore than 92% of Acinetobacter baumannii collected were multidrug-resistant (MDR) and/or carbapenem-resistant (CR), and > 50% susceptibility was reported only to minocycline (62.2% among both MDR and CR isolates). Rates of MDR and CR Pseudomonas aeruginosa were 14.3% and 20.5%, respectively, and among all P. aeruginosa collected from adults, susceptibility to ceftazidime/avibactam was 95.3% and to ceftolozane/tazobactam was 88.4%. For Escherichia coli from adults and MDR E. coli, susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam, imipenem, meropenem and meropenem/vaborbactam was 92.1%–98.7%. Susceptibility to tigecycline was > 94% among Klebsiella pneumoniae from adult, paediatric, and ICU patients (all ages). CTX-M-15 was the most frequently identified β-lactamase gene among E. coli and K. pneumoniae. Susceptibility to most antimicrobial agents was < 50% among K. pneumoniae carrying CTX-M-15, CTX-M-9-type, NDM-5, and/or OXA-48 β-lactamase genes. All S. aureus collected were susceptible to teicoplanin, vancomycin, daptomycin, linezolid and tigecycline, with 96.1% of S. aureus from adults were susceptible to ceftaroline. Overall, 58.8% of Staphylococcus aureus were MRSA.ConclusionThis study provides valuable information regarding antimicrobial susceptibility in Jordan between 2010 and 2021. Continued monitoring of in vitro antimicrobial susceptibility is critical in the fight against antimicrobial resistance.

Abstract Background Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA – Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS). Results We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently. Conclusion Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define.

Background: Antimicrobial resistance is a serious concern and life threat in public health globally. Gram-positive clinical isolates are one of the leading etiological agents showing drug resistance traits. This study aimed to detect the antimicrobial resistance pattern of clinically isolated Gram-positive bacterial isolates collected from various renowned diagnostic centers of Dhaka city, Bangladesh. Methods: A laboratory-based cross-sectional study was conducted at the Microbiological Laboratory of the Department of Microbiology, Stamford University Bangladesh. A total of 104 clinical specimens (wound swab, pus, blood, urine, ear discharge, nasal swab, sputum, nipple discharge, breast milk, catheter tip, umbilical swab, and throat swab) from different age groups were collected from some renowned diagnostic centers of Dhaka city for analyzing the cultural and drug sensitivity patterns from January 2023 to April 2023. According to the study plan, only the Gram-positive bacteria were collected and transported to the laboratory of the Department of Microbiology, Stamford University Bangladesh following WHO guidelines, and the antimicrobial susceptibility tests were carried out by Kirby–Bauer disk diffusion method and the results were interpreted as per CLSI guidelines, 2022. Results: This study highlights the presence of Staphylococcus aureus (65.4%), Streptococcus spp. (23.1%), and Enterococcus spp. (11.5%) as causative agents of Gram-positive bacterial infections from overall 104 clinical specimens. Most of our isolates were found multidrug-resistant. For S. aureus isolates, cefixime (100%); for Streptococcus spp., doxycycline (95.83%); and for Enterococcus spp., amoxicillin (100%), cefixime (100%), colistin (100%), and cotrimoxazole (100%) were found to be the most ineffective drug when compared to other antibiotics. Conclusion: Our study portrayed the remarkable antibiotic resistance feature of Gram-positive bacterial isolates which is a matter of public health concern. It is necessary to carry out a continuous assessment of antibiotic sensitivity patterns of resistant Gram-positive bacteria; otherwise, it will become an uncontrollable problem not only in Bangladesh but also worldwide in the near future.

Ailong Huang,&ast; Yuchao Wang,&ast; Haiyan Xu, Xiuxiu Jin, Bingqing Yan, Wei Zhang Department of Microbiology, Nantong Center for Disease Control and Prevention, Nantong, Jiangsu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Wei Zhang, Department of Microbiology, Nantong Center for Disease Control and Prevention, No. 189, Gongnong South Road, Chongchuan District, Nantong City, Jiangsu Province, People’s Republic of China, Email zhangwei_1886@sina.comPurpose: The objective of this study was to investigate the prevalence and molecular characteristics of Vibrio parahaemolyticus isolates from fecal samples of patients in Nantong, China.Methods: From 2018 to 2021, a total of 106 clinical cases and samples of V. parahaemolyticus infection were collected. The virulence genes, serotypes and antibiotic resistance of these isolates were analyzed. Additionally, pulsed-field gel electrophoresis (PFGE) was used to analyze the homogeneity of the isolates.Results: Outbreaks of V. parahaemolyticus infection were concentrated in the summer, with seafood consumption being the primary contributing factor, followed by meat and meat products. tlh+tdh+trh- was confirmed as the most frequently detected virulence genotype among the clinical isolates. 16 serotypes were identified, and O3:K6 was the dominant serotype in Nantong. The antimicrobial susceptibility testing revealed the highest resistance rate to cefazolin (99.1%, 104/106), followed by ampicillin (64.2%, 68/106) and tetracycline (29.2%, 31/106). Fourteen resistant phenotypes were identified, with ampicillin-cefazolin being the most prevalent. The multiple antibiotic resistance (MAR) index ranged from 0.07 to 0.36. PFGE typing clustered isolates with similarity greater than 85% into ten genetic clusters (A-J).Conclusion: Clinical isolates generally exhibited pathogenicity and drug resistance, with some isolates displaying high homology. Clusters C, E, and G were the predominant circulating clusters in this area, posing a potential risk of recurrent outbreaks, which demanded our vigilance.Keywords: Vibrio parahaemolyticus, clinical isolates, antibiotic resistance, PFGE

Onychomycosis is a common fungal infection that affects the nails and accounts for approximately 50% of all nail diseases. The main pathogens involved include dermatophytes, such as Trichophyton rubrum, members of the T. mentagrophytes complex, and emerging pathogens in this infection, T. schoenleinii and T. tonsurans. Tea tree (Melaleuca alternifolia Cheel) essential oil (EO) has been proposed as a promising natural alternative to traditional treatments due to its antimicrobial properties. Among its more than 100 compounds, terpinen-4-ol is one of the main contributors to the antifungal action of this EO. To determine the antifungal activity of tea tree EO against dermatophytes, we designed an in vitro study using EUCAST-AFST protocols to obtain the values of MIC (minimum inhibitory concentration) and MFC (minimum fungicidal concentration) of several commercial M. alternifolia Cheel EOs against three species of dermatophytes isolated from clinical samples with suspected toenail onychomycosis. The results showed that the microorganism most sensitive to the action of the EO was T. rubrum, which had an MIC value more than 13 times lower than the value obtained for T. schoenleinii (0.4% v/v), the most resistant isolate. No differences in antifungal activity were observed by the analysed EOs or between the MIC and MFC values. These in vitro results suggest that tea tree EO is a viable option for the alternative treatment of onychomycosis, although clinical studies are needed to confirm the long-term antifungal activity, safety and efficacy of the oils studied in a clinical context.

Selective forces in the environment drive bacterial adaptation to novel niches, choosing the fitter variants in the population. However, in dynamic and changing environments, the evolutionary processes controlling bacterial adaptation are difficult to monitor. Here, we follow 9 people with cystic fibrosis chronically infected with Pseudomonas aeruginosa , as a proxy for bacterial adaptation. We identify and describe the bacterial changes and evolution occurring between 15 and 35 yr of within-host evolution. We combine whole-genome sequencing, RNA sequencing, and metabolomics and compare the evolutionary trajectories directed by the adaptation of 4 different P. aeruginosa lineages to the lung. Our data suggest divergent evolution at the genomic level for most of the genes, with signs of convergent evolution with respect to the acquisition of mutations in regulatory genes, which drive the transcriptional and metabolomic program at late time of evolution. Metabolomics further confirmed convergent adaptive phenotypic evolution as documented by the reduction of the quorum-sensing molecules acyl-homoserine lactone, phenazines, and rhamnolipids (except for quinolones). The modulation of the quorum-sensing repertoire suggests that similar selective forces characterize at late times of evolution independent of the patient. Collectively, our data suggest that similar environments and similar P. aeruginosa populations in the patients at prolonged time of infection are associated with an overall reduction of virulence-associated features and phenotypic convergence. [ABSTRACT FROM AUTHOR]

Background: Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA – Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS). Results: We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently. Conclusion: Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define. [ABSTRACT FROM AUTHOR]

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10–15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future. [ABSTRACT FROM AUTHOR]

IntroductionMycoplasma hominis (M. hominis) belongs to the class Mollicutes, characterized by a very small genome size, reduction of metabolic pathways, including transcription factors, and the absence of a cell wall. Despite this, they adapt well not only to specific niches within the host organism but can also spread throughout the body, colonizing various organs and tissues. The adaptation mechanisms of M. hominis, as well as their regulatory pathways, are poorly understood. It is known that, when adapting to adverse conditions, Mycoplasmas can undergo phenotypic switches that may persist for several generations.MethodsTo investigate the adaptive properties of M. hominis related to survival in the host, we conducted a comparative phenotypic and proteogenomic analysis of eight clinical isolates of M. hominis obtained from patients with urogenital infections and the laboratory strain H-34.ResultsWe have shown that clinical isolates differ in phenotypic features from the laboratory strain, form biofilms more effectively and show resistance to ofloxacin. The comparative proteogenomic analysis revealed that, unlike the laboratory strain, the clinical isolates possess several features related to stress survival: they switch carbon metabolism, activating the energetically least advantageous pathway of nucleoside utilization, which allows slowing down cellular processes and transitioning to a starvation state; they reconfigure the repertoire of membrane proteins; they have integrative conjugative elements in their genomes, which are key mediators of horizontal gene transfer. The upregulation of the methylating subunit of the restriction-modification (RM) system type I and the additional components of RM systems found in clinical isolates suggest that DNA methylation may play a role in regulating the adaptation mechanisms of M. hominis in the host organism. It has been shown that based on the proteogenomic profile, namely the genome sequence, protein content, composition of the RM systems and additional subunits HsdM, HsdS and HsdR, composition and number of transposable elements, as well as the sequence of the main variable antigen Vaa, we can divide clinical isolates into two phenotypes: typical colonies (TC), which have a high growth rate, and atypical (aTC) mini-colonies, which have a slow growth rate and exhibit properties similar to persisters.DiscussionWe believe that the key mechanism of adaptation of M. hominis in the host is phenotypic restructuring, leading to a slowing down cellular processes and the formation of small atypical colonies. This is due to a switch in carbon metabolism and activation the pathway of nucleoside utilization. We hypothesize that DNA methylation may play a role in regulating this switch.

Carbapenem-resistant Acinetobacter spp. is associated with nosocomial infections in intensive care unit patients, resulting in high mortality. Although Acinetobacter spp. represent a serious public health problem worldwide, there are a few studies related to the presence of carbapenemases in health care facilities and other environmental settings in Ecuador. The main aim of this study was to characterize the carbapenem-resistant Acinetobacter spp. isolates obtained from four hospitals (52) and from five rivers (27) close to Quito. We used the disc diffusion and EDTA sinergy tests to determine the antimicrobial susceptibility and the production of metallo β-lactamases, respectively. We carried out a multiplex PCR of gyrB gene and the sequencing of partial rpoB gene to bacterial species identification. We performed molecular screening of nine carbapenem-resistant genes (blaSPM, blaSIM, blaGIM, blaGES, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, and blaOXA-143) by multiplex PCR, followed by identification using sequencing of blaOXA genes. Our findings showed that carbapenem-resistant A. baumannii were the main species found in health care facilities and rivers. Most of the clinical isolates came from respiratory tract samples and harbored blaOXA-23, blaOXA-366, blaOXA-72, blaOXA-65, blaOXA-70, and blaOXA-143-like genes. The river isolates harbored only the blaOXA-51 and probably blaOXA-259 genes. We concluded that the most predominant type of carbapenem genes among isolates were both blaOXA-23 and blaOXA-65 among A. baumannii clinical isolates.

IntroductionEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. are microorganisms referred as the ESKAPE group pathogens. These microorganisms have generated great concern in health institutions around the world since most of them have resistance to multiple antibiotics and cause most infections associated with healthcare, as well as community infections. The aim of this study was the analysis of antibiotic resistance in microorganisms of the ESKAPE group, recovered from clinical samples in 11 health institutions from Hermosillo and Ciudad Obregón in the State of Sonora, México, during the period from 2019 to 2020.MethodsA cross-sectional, descriptive, observational, and temporality epidemiological study was carried out. A comparative and statistical analysis of antibiotic resistance was carried out using the chi-square test, and small values were analyzed using Fisher’s exact test p ≤ 0.05.Results and discussionAll the ESKAPE group microorganisms showed significant differences in antibiotic resistance percentages between both cities. High resistance percentages for some antibiotics, like cephalosporins and ciprofloxacin were detected for Klebsiella pneumoniae and Acinetobacter baumannii.

Martin Rihacek,1 Michaela Kuthanova,1 Zbynek Splichal,1 Vojtech Adam,1 Kristyna Hrazdilova,1 Radek Vesely,2,3 Ludek Zurek,1 Kristyna Cihalova1 1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic; 2Department of Traumatology at the Medical Faculty, Masaryk University and Trauma Hospital Brno, Brno, Czech Republic; 3Department of Traumatology, Faculty of Medicine, Masaryk University, Brno, Czech RepublicCorrespondence: Kristyna Cihalova, Department of Chemistry and Biochemistry, Mendel University in Brno, Zem&ecaron;d&ecaron;lská 1, Brno, CZ-613 00, Czech Republic, Tel +420-5-4513-3350, Fax +420-5-4521-2044, Email kriki.cihalova@seznam.czPurpose: Resistance of pathogenic strains of Escherichia coli to β-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to β-lactams.Materials and Methods: Twelve E. coli isolates obtained from patients’ wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected β-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of β-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin.Results: Phenotypic resistance to β-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin.Conclusion: Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.Keywords: clinical isolates, ampicillin resistance, resistance mechanisms, antibiotics

Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.

Abstract Background Stenotrophomonas maltophilia is able to cause infections in immunocompromised patients, and the treatment of this opportunistic pathogen is complicated due to its virulence factors, antibiotic resistance, and the ability of the bacteria to produce biofilm. The main goals of this study were to assess the susceptibility of extensively drug-resistant (XDR) isolates to ethanol and EDTA, and evaluating the synergistic effect of these disinfectants, and also survey the effect of exposure to sub-inhibitory concentrations of ethanol and EDTA on the expression of biofilm-producing smf-1, rpfF genes. Results The results showed that EDTA significantly increased the effectiveness of the ethanol and have a synergistic effect. All of the 10 XDR isolates included in the current study harbored smf-1 and rpfF genes and produced biofilm. After exposure to MIC, sub-MIC, synergism, and sub-synergism of ethanol and EDTA, the expression of smf-1 and rpfF genes was repressed significantly. Conclusion In the current study, it was indicated that the expression of biofilm-producing genes was repressed when bacteria are exposed to different concentrations of ethanol and EDTA. Future studies should include more complex microbial communities residing in the hospitals, and more disinfectants use in hospitals. Expression of other virulence genes in different conditions is suggested.

Pseudomonas aeruginosa, a gram-negative bacterium, is notorious for its innate resistance to many antibiotics. Carbapenems are broad-spectrum antibiotics often used to treat severe P. aeruginosa infections. However, the emergence and proliferation of carbapenem-resistant P. aeruginosa (CRPA) strains have become a grave global health concern. This study examined the co-occurrence of four major carbapenemase genes, namely blaNDM, blaVIM, blaKPC, and blaOXA-48, in clinical isolates of P. aeruginosa. Using standard microbiological methods,150 P. aeruginosa clinical isolates were collected and identified, and antimicrobial susceptibility testing was conducted following Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) with gene-specific primers was used to detect the presence of carbapenemase genes. Among the 150 P. aeruginosa clinical isolates, 62(41.3%) were found to be carbapenem-resistant. The most detected carbapenemase genes were blaKPC (49%), blaNDM (31%), blaOXA-48 (22%), and blaVIM (9%). Notably 13(12.9%) isolates carried two carbapenemase genes. The combination of blaKPC and blaNDM genes was found in eight isolates, two isolates carried blaKPC and blaVIM, and three isolates carried blaOXA-48 and blaNDM. Four isolates (6.5%) harbored three carbapenemase genes. Co-occurrence of blaNDM, blaVIM, blaKPC, and blaOXA-48 was observed in four isolates (2.8%). Our findings highlight the alarming prevalence of carbapenemase genes, particularly blaNDM and blaKPC, in clinical isolates of P. aeruginosa. The co-occurrence of multiple carbapenemase genes in the same isolate raises concerns about the potential for horizontal gene transfer and dissemination of multidrug-resistant P. aeruginosa strains in clinical settings. Further research is needed to elucidate the molecular mechanisms underlying the co-occurrence of carbapenemase genes and their impact on the clinical outcomes of P. aeruginosa infections. Urgent measures, such as enhanced surveillance, infection control protocols, and antibiotic stewardship programs, are imperative to combat the emergence and spread of CRPA strains.

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings.

Although cases of Legionnaires’ disease are notifiable, data on the phenotypic and genotypic characterisation of clinical isolates are limited. This retrospective study aims to report the results of the characterisation of Legionella clinical isolates in Spain from 2012 to 2022. Monoclonal antibodies from the Dresden panel were used for phenotypic identification of Legionella pneumophila. Genotypic characterisation and sequence type assignment were performed using the Sequence-Based Typing scheme. Of the 1184 samples, 569 were identified as Legionella by culture. Of these, 561 were identified as L. pneumophila, of which 521 were serogroup 1. The most common subgroups were Philadelphia (n = 107) and Knoxville (n = 106). The SBT analysis revealed 130 different STs, with the most common genotypes being ST1 (n = 87), ST23 (n = 57), ST20 (n = 30), and ST42 (n = 29). Knoxville has the highest variability with 32 different STs. ST23 is mainly found in Allentown/France (n = 46) and ST42 in Benidorm (n = 18), whereas ST1 is widely distributed. The results demonstrate that clinical isolates show high genetic diversity, although only a few sequence types (STs) are responsible for most cases. However, outbreaks can also occur with rare genotypes. More data on LD and associated epidemiological studies are needed to establish the risk of an isolate causing outbreak in the future.

Pseudomonas aeruginosa, Staphylococcus aureus, and Burkholderia cepacia are notorious pathogens known for their ability to form resilient biofilms, particularly within the lung environment of cystic fibrosis (CF) patients. The heightened concentration of NaCl, prevalent in the airway liquid of CF patients' lungs, has been identified as a factor that promotes the growth of osmotolerant bacteria like S. aureus and dampens host antibacterial defenses, thereby fostering favorable conditions for infections.In this study, we aimed to investigate how increased NaCl concentrations impact the development of multi-species biofilms in vitro, using both laboratory strains and clinical isolates of P. aeruginosa, S. aureus, and B. cepacia co-cultures. Employing a low-nutrient culture medium that fosters biofilm growth of the selected species, we quantified biofilm formation through a combination of adherent CFU counts, qPCR analysis, and confocal microscopy observations.Our findings reaffirmed the challenges faced by S. aureus in establishing growth within 1:1 mixed biofilms with P. aeruginosa when cultivated in a minimal medium. Intriguingly, at an elevated NaCl concentration of 145 mM, a symbiotic relationship emerged between S. aureus and P. aeruginosa, enabling their co-existence. Notably, this hyperosmotic environment also exerted an influence on the interplay of these two bacteria with B. cepacia. We demonstrated that elevated NaCl concentrations play a pivotal role in orchestrating the distribution of these three species within the biofilm matrix.Furthermore, our study unveiled the beneficial impact of NaCl on the biofilm growth of clinically relevant mucoid P. aeruginosa strains, as well as two strains of methicillin-sensitive and methicillin-resistant S. aureus. This underscores the crucial role of the microenvironment during the colonization and infection processes. The results suggest that hyperosmotic conditions could hold the key to unlocking a deeper understanding of the genesis and behavior of CF multi-species biofilms.

Introduction: The rise of multidrug-resistant (MDR) microorganisms, which pose a grave threat, has made choosing antibiotics to treat bacterial infections incredibly challenging. The prescription antibiotics have to consistently be effective against the identified related infections. Thus the research team wanted to find the antibiotic sensitivity profiles and pathogenic bacterial isolates in various patient specimens. Methods: The collection of 403 clinical samples of throat swabs, sputum, blood, stool, and urine from people of both genders and various ages was performed aseptically. Identification was carried out by microscopic, cultural, biochemical, and serological analysis. Finally, the disk diffusion method by Kirby-Bauer was employed to determine antibiotic responsiveness profiling. Results: A total of 93 samples (23.08%) were identified as positive isolates comprising 28.74% (25/87) of urine culture, followed by 24.32% (27/111) of blood, 23.08% (15/65) of stool, 20% (07/35) of throat swabs, and 18.1% (19/105) of sputum culture. Gram-negative bacteria 81(87.1%) showed more prevalence than Gram-positive bacteria 12 (12.90%). The most frequently identified isolates were Klebsiella spp. (24%), Salmonella spp. (23%), E. coli (17%), Pseudomonas spp. (5%), and Enterococcus spp. (3%). The infections showed more prevalence within the age group of 16-46 years (60.2%) and among males (48%) than females (46%). Conclusions: It was found that E. coli was the most abundant bacteria isolated from urine, Klebsiella spp. was the most abundant bacteria isolated from sputum and throat swabs, and Shiga toxin-producing Escherichia coli was the most abundant bacteria isolated from stool. Amikacin, Ciprofloxacin, Gentamicin, Imipenem, and Levofloxacin were considerably effective antibiotics, whereas Cefotaxime, Ceftazidime, Cefuroxime, Mecillinam, and Meropenem were least effective. [ABSTRACT FROM AUTHOR]

Introduction: The emergence of carbapenem-resistant Enterobacteriaceae (CRE) poses a significant public health concern due to its potential for increased mortality and morbidity. The limited availability of effective antibiotics further exacerbates the dissemination of carbapenem-resistant bacteria. This study aimed to evaluate the prevalence of carbapenem resistance and carbapenemase production in Enterobacteriaceae isolates using the Modified Hodge Test. Methods: This observational study was conducted at the Department of Microbiology, MGM Medical College & Hospital, Aurangabad, Maharashtra, from November 2015 to November 2017. 171 Enterobacteriaceae isolates from various clinical samples were comprehensively tested for carbapenem resistance and carbapenemase production. This involved the use of carbapenem discs (ertapenem, meropenem, and imipenem), E-test strips for ertapenem and meropenem, and the Modified Hodge Test (MHT) for carbapenemase identification. Results: Among the 171 tested Enterobacteriaceae isolates, a substantial proportion (40%) displayed resistance to carbapenems, as determined by disc diffusion and E-test methods. Among the carbapenem-resistant isolates, 13 were positive for the MHT. Conclusion: Our study revealed a notable prevalence of carbapenem resistance in Enterobacteriaceae isolates from a tertiary care hospital. The MHT, following Clinical and Laboratory Standards Institute (CLSI) guidelines, demonstrated high sensitivity (> 90%) and specificity (> 90%) for detecting KPC-type carbapenemases in these isolates. Treatment options for CRE infections are limited, with tigecycline and colistin identified as potential options. Our study highlights the importance of promptly diagnosing different carbapenemases using PCR techniques. Consequently, we strongly advocate for implementing robust antimicrobial stewardship programs and infection control practices in healthcare settings to prevent CRE spread effectively. [ABSTRACT FROM AUTHOR]

Antimicrobial drug resistance has made the treatment of microbial infections quite challenging. A Gram-positive, anaerobic, spore-forming, and toxin-producing bacillus, Clostridioides difficile infection causes diarrhea-related deaths globally. The available drugs like vancomycin and metronidazole are becoming less effective against this infection. We have designed this study to identify genes responsible for antimicrobial resistance and have a better understanding of the mutations and their impact on the antimicrobial resistance activity. The Whole Genome Sequencing data of 11 C. difficile clinical isolates was analyzed to determine novel genes playing a significant role in antimicrobial resistance mechanisms. Comparative structure analysis of wild and mutant structures of proteins and their functions provided insight into the impact of the identified mutations on antimicrobial resistance. We identified 8 genes common in all the isolates that play a vital role in drug resistance through antibiotic efflux, ribosomal protection, and antibiotic inactivation. Variations in the functional domains of tetA(P), tetM, and ermB genes were found to be the most promising novel drug targets. Our findings suggest that these novel gene mutations would be beneficial in designing new drugs to combat C. difficile infection. [ABSTRACT FROM AUTHOR]

The heterogenicity of antimicrobial resistance genes described in clinically significant bacterial isolates and their potential role in reducing the efficacy of classically effective antibiotics pose a major challenge for global healthcare, especially in infections caused by Gram-negative bacteria. We analyzed 112 multidrug-resistant (MDR) isolates from clinical samples in order to detect high resistance profiles, both phenotypically and genotypically, among four Gram-negative genera (Acinetobacter, Escherichia, Klebsiella, and Pseudomonas). We found that 9.8% of the total selected isolates were classified as extensively drug-resistant (XDR) (six isolates identified as A. baumannii and five among P. pneumoniae isolates). All other isolates were classified as MDR. Almost 100% of the isolates showed positive results for blaOXA-23 and blaNDM-1 genes among the A. baumannii samples, one resistance gene (blaCTX-M) among E. coli, and two genetic determinants (blaCTX-M and aac(6′)-Ib) among Klebsiella. In contrast, P. aeruginosa showed just one high-frequency antibiotic resistance gene (dfrA), which was present in 68.42% of the isolates studied. We also describe positive associations between ampicillin and cefotaxime resistance in A. baumannii and the presence of blaVEB and blaGES genes, as well as between the aztreonam resistance phenotype and the presence of blaGES gene in E. coli. These data may be useful in achieving a better control of infection strategies and antibiotic management in clinical scenarios where these multidrug-resistant Gram-negative pathogens cause higher morbidity and mortality.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 μg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 μg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

The opportunistic fungus Candida albicans is the leading cause of invasive candidiasis in immune-compromised individuals. Drugs from the echinocandin (ECN) class, including caspofungin, are used as a first line of therapy against invasive candidiasis. The only known mechanism of clinical resistance to ECNs is point mutations in the FKS1 gene, which encodes the drug target. However, many clinical isolates developed decreased ECN susceptibilities in the absence of resistance-associated FKS1 mutations. We have identified 15 C. albicans genes that contribute to decreased drug susceptibility. We explored the expression of these 15 genes in clinical isolates with different levels of ECN susceptibility. We found that these 15 genes are expressed in clinical isolates with or without FKS1 mutations, including those strains that are less susceptible to ECNs. In addition, FKS1 expression was increased in such less susceptible isolates compared to highly susceptible isolates. Similarities of gene expression patterns between isolates with decreased ECN susceptibilities in the absence of FKS1 mutations and clinically resistant isolates with mutations in FKS1 suggest that clinical isolates with decreased ECN susceptibilities may be a precursor to development of resistance.

ABSTRACT Our overall understanding of the developmental biology of malaria parasites has been greatly enhanced by recent advances in transcriptomic analysis. However, most of these investigations rely on laboratory strains (LS) that were adapted into in vitro culture many years ago, and the transcriptomes of clinical isolates (CI) circulating in human populations have not been assessed. In this study, RNA-seq was used to compare the global transcriptome of mid-stage gametocytes derived from three short-term cultured CI, with gametocytes derived from the NF54 reference laboratory strain. The core transcriptome appeared to be consistent between CI- and LS-derived gametocyte preparations, but some important differences were also observed. A majority of gametocyte-specific genes (43/53) appear to have relatively higher expression in CI-derived gametocytes than in LS-derived gametocytes, but a K-means clustering analysis showed that genes involved in flagellum- and microtubule-based processes (movement/motility) were more abundant in both groups, albeit with some differences between them. In addition, gametocytes from one CI described as CI group II gametocytes (CI:GGII) showed gene expression variation in the form of reduced gametocyte-specific gene expression compared to the other two CI-derived gametocytes (CI gametocyte group I, CI:GGI), although the mixed developmental stages used in our study is a potential confounder, only partially mitigated by the inclusion of multiple replicates for each CI. Overall, our study suggests that there may be subtle differences in the gene expression profiles of mid-stage gametocytes from CI relative to the NF54 reference strain of Plasmodium falciparum. Thus, it is necessary to deploy gametocyte-producing clinical parasite isolates to fully understand the diversity of gene expression strategies that may occur during the sequestered development of parasite sexual stages. IMPORTANCE Maturing gametocytes of Plasmodium falciparum are known to sequester away from peripheral circulation into the bone marrow until they are mature. Blocking gametocyte sequestration can prevent malaria transmission from humans to mosquitoes, but most studies aim to understand gametocyte development utilizing long-term adapted laboratory lines instead of clinical isolates. This is a particular issue for our understanding of the sexual stages, which are known to decrease rapidly during adaptation to long-term culture, meaning that many LS are unable to produce transmissible gametocytes. Using RNA-seq, we investigated the global transcriptome of mid-stage gametocytes derived from three clinical isolates and a reference strain (NF54). This identified important differences in gene expression profiles between immature gametocytes of CI and the NF54 reference strain of P. falciparum, suggesting increased investment in gametocytogenesis in clinical isolates. Our transcriptomic data highlight the use of clinical isolates in studying the morphological, cellular features and molecular biology of gametocytes.