Australian Group on Antimicrobial Resistance (AGAR) Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP) Bloodstream Infection Annual Report 2022.

From 1 January to 31 December 2022, fifty-five institutions across Australia participated in the Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP). The aim of ASSOP 2022 was to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that were antimicrobial resistant, with particular emphasis on susceptibility to methicillin and on characterisation of the molecular epidemiology of the methicillin-resistant isolates. A total of 3,214 SAB episodes were reported, of which 77.5% were community-onset. Overall, 15.0% of S. aureus were methicillin resistant. The 30-day all-cause mortality associated with methicillin-resistant SAB was 21.4%, which was significantly different to the 16.8% all-cause mortality associated with methicillin-susceptible SAB (p = 0.02). With the exception of the ß-lactams and erythromycin, antimicrobial resistance in methicillin-susceptible S. aureus was rare. However, in addition to the ß-lactams, approximately 31% of methicillin-resistant S. aureus (MRSA) were resistant to ciprofloxacin; 30% to erythromycin; 13% to tetracycline; 11% to gentamicin; and 2% to co-trimoxazole. One MRSA isolate, with a daptomycin MIC of 1.5 mg/L, harboured the A302V mprF and A23V cls2 mutations. When applying the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, teicoplanin resistance was detected in one MRSA isolate. Resistance to vancomycin or linezolid was not detected. Resistance to non-ß-lactam antimicrobials was largely attributable to the healthcare-associated MRSA (HA-MRSA) clone ST22-IV [2B] (EMRSA-15), and to the community-associated MRSA (CA-MRSA) clone ST45-V [5C2&5] which has acquired resistance to multiple antimicrobials including ciprofloxacin, clindamycin, erythromycin, gentamicin, and tetracycline. The ST22-IV [2B] (EMRSA-15) clone is the predominant HA-MRSA clone in Australia. Nonetheless, 86% of methicillin-resistant SAB episodes were due to CA-MRSA clones. Although polyclonal, approximately 72% of CA-MRSA clones were characterised as ST93-IV [2B] (Queensland clone); ST5-IV [2B]; ST45-V [5C2&5]; ST1-IV [2B]; ST30-IV [2B]; ST97-IV [2B]; ST953-IV [2B]; and ST8-IV [2B]. As CA-MRSA is well established in the Australian community, it is important to monitor antimicrobial resistance patterns in community- and healthcare-associated SAB as this information will guide therapeutic practices in treating S. aureus bacteraemia.

[Comparison of Epsilometer test and agar dilution method in detecting the sensitivity of Helicobacter pylori to metronidazole].

OBJECTIVE: Agar dilution method (ADM) was used as the golden standard to evaluate the consistency of Epsilometer test (E-test) in detecting the sensitivity of Helicobacter pylori (H. pylori) to metronidazole. METHODS: From August 2018 to July 2020, patients with H. pylori infection treated for the first time in Peking University Third Hospital for gastroscopy due to dyspepsia were included in this study. Gastric mucosas were taken from the patients with H. pylori infection. H. pylori culture was performed. Both the ADM and E-test were applied to the antibiotic susceptibility of H. pylori to metro-nidazole, and the consistency and correlation between the two methods were validated. RESULTS: In the study, 105 clinical isolates of H. pylori were successfully cultured, and the minimum inhibitory concentration ≥ 8 mg/L was defined as drug resistance. Both ADM and the E-test showed high resistance rates to metronidazole, 64.8% and 62.9%, respectively. Among them, 66 drug-resistant strains were detected by ADM and E-test, and 37 were sensitive strains, so the consistency rate was 98.1%. Two strains were evaluated as drug resistance by ADM, but sensitive by the E-test, with a very major error rate of 1.9%. There was zero strain sensitive according to ADM but assessed as resistant by the E-test, so the major error rate was 0%. Taking ADM as the gold standard, the sensitivity of E-test in the detection of metronidazole susceptibility was 97.1% (95%CI: 0.888-0.995), and the specificity was 100% (95%CI: 0.883-1.000). Cohen's kappa analysis showed substantial agreement, and kappa coefficient was 0.959 (95%CI: 0.902-1.016, P < 0.001). Spearmans correlation analysis confirmed this correlation was significant (r=0.807, P < 0.001). The consistency evaluation of Bland-Altman method indicated that it was good, and there was no measured value outside the consistency interval. In this study, cost analysis, including materials and labor, showed a 32.2% higher cost per analyte for ADM as compared with the E-test (356.6 yuan vs. 269.8 yuan). CONCLUSION: The susceptibility test of H. pylori to metronidazole by E-test presents better agreement with ADM. Because it is less expensive, less labor intensive, and more rapid, it is an easy and reliable method for H. pylori susceptibility testing.

Reliable and rapid identification of terbinafine resistance in dermatophytic nail and skin infections.

BACKGROUND: Fungal infections are the most frequent dermatoses. The gold standard treatment for dermatophytosis is the squalene epoxidase (SQLE) inhibitor terbinafine. Pathogenic dermatophytes resistant to terbinafine are an emerging global threat. Here, we determine the proportion of resistant fungal skin infections, analyse the molecular mechanisms of terbinafine resistance, and validate a method for its reliable rapid identification. METHODS: Between 2013 and 2021, we screened 5634 consecutively isolated Trichophyton for antifungal resistance determined by hyphal growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine. All Trichophyton isolates with preserved growth capacity in the presence of terbinafine underwent SQLE sequencing. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. RESULTS: Over an 8-year period, the proportion of fungal skin infections resistant to terbinafine increased from 0.63% in 2013 to 1.3% in 2021. Our routine phenotypic in vitro screening analysis identified 0.83% (n = 47/5634) of Trichophyton strains with in vitro terbinafine resistance. Molecular screening detected a mutation in the SQLE in all cases. Mutations L393F, L393S, F397L, F397I, F397V, Q408K, F415I, F415S, F415V, H440Y, or A398 A399 G400 deletion were detected in Trichophyton rubrum. Mutations L393F and F397L were the most frequent. In contrast, all mutations detected in T. mentagrophytes/T. interdigitale complex strains were F397L, except for one strain with L393S. All 47 strains featured significantly higher MICs than terbinafine-sensitive controls. The mutation-related range of MICs varied between 0.004 and 16.0 µg/mL, with MIC as low as 0.015 µg/mL conferring clinical resistance to standard terbinafine dosing. CONCLUSIONS: Based on our data, we propose MIC of 0.015 µg/mL as a minimum breakpoint for predicting clinically relevant terbinafine treatment failure to standard oral dosing for dermatophyte infections. We further propose growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine and SQLE sequencing as fungal sporulation-independent methods for rapid and reliable detection of terbinafine resistance.

Polyphenylene carboxymethylene (PPCM), the active component of the topical contraceptive Yaso-GEL, exhibits potent antimicrobial activity against Neisseria gonorrhoeae in preclinical studies.

INTRODUCTION: Polyphenylene carboxymethylene (PPCM) is a condensation polymer that has both contraceptive and antimicrobial activity against several sexually transmitted viruses including HIV, herpes simplex virus, Ebola virus and SARS-CoV-2 in preclinical studies. PPCM, both as an active pharmaceutical ingredient (API) and in a vaginal gel formulation (Yaso-GEL), has an excellent safety profile. Here, we evaluated the efficacy of PPCM against Neisseria gonorrhoeae in vitro and in a gonorrhoea mouse model. METHODS: The minimal inhibitory concentration (MIC) of PPCM was determined against 11 N. gonorrhoeae strains by agar dilution and a microtitre plate-based method. In vivo efficacy was tested in a murine model of N. gonorrhoeae genital tract infection by applying Yaso-GEL, PPCM incorporated in 2.7% hydroxyethylcellulose (HEC), or the HEC vehicle vaginally prior to challenge with N. gonorrhoeae. Vaginal swabs were quantitatively cultured over 5 days to assess efficacy. RESULTS: PPCM MIC against N. gonorrhoeae ranged between 5-100 µg/mL (agar dilution) and 50-200 µg/mL (microtitre plate method). PPCM/HEC gel applied vaginally prior to bacterial challenge resulted in a concentration-dependent inhibition of infection. Yaso-GEL containing 4% PPCM prevented infection in 100% of mice. Incubation of N. gonorrhoeae with PPCM increased membrane permeability, suggesting PPCM directly compromises N. gonorrhoeae viability, which may be a mechanism by which PPCM inhibits N. gonorrhoeae infection. CONCLUSIONS: Yaso-GEL containing the API PPCM showed significant activity against N. gonorrhoeae in vitro and in vivo in a female mouse model. These data support further development of Yaso-GEL as an inexpensive, non-hormonal and non-systemic product with both contraceptive and antimicrobial activity against gonorrhea and other common sexually transmitted infections (STIs). Such multipurpose prevention technology products are needed by women in all economic, social and cultural circumstances to prevent unintended pregnancy and STIs.

Bacterial pathogens in pediatric appendicitis: a comprehensive retrospective study.

Background: Appendicitis is a frequent condition, with peak incidences in the second decade of life. Its pathogenesis is under debate, but bacterial infections are crucial, and antibiotic treatment remains essential. Rare bacteria are accused of causing complications, and various calculated antibiotics are propagated, yet there is no comprehensive microbiological analysis of pediatric appendicitis. Here we review different pre-analytic pathways, identify rare and common bacterial pathogens and their antibiotic resistances, correlate clinical courses, and evaluate standard calculated antibiotics in a large pediatric cohort. Method: We reviewed 579 patient records and microbiological results of intraoperative swabs in standard Amies agar media or fluid samples after appendectomies for appendicitis between May 2011 and April 2019. Bacteria were cultured and identified via VITEK 2 or MALDI-TOF MS. Minimal inhibitory concentrations were reevaluated according to EUCAST 2022. Results were correlated to clinical courses. Results: Of 579 analyzed patients, in 372 patients we got 1330 bacterial growths with resistograms. 1259 times, bacteria could be identified to species level. 102 different bacteria could be cultivated. 49% of catarrhal and 52% of phlegmonous appendices resulted in bacterial growth. In gangrenous appendicitis, only 38% remained sterile, while this number reduced to 4% after perforation. Many fluid samples remained sterile even when unsterile swabs had been taken simultaneously. 40 common enteral genera were responsible for 76.5% of bacterial identifications in 96.8% of patients. However, 69 rare bacteria were found in 187 patients without specifically elevated risk for complications. Conclusion: Amies agar gel swabs performed superior to fluid samples and should be a standard in appendectomies. Even catarrhal appendices were only sterile in 51%, which is interesting in view of a possible viral cause. According to our resistograms, the best in vitro antibiotic was imipenem with 88.4% susceptible strains, followed by piperacillin-tazobactam, cefuroxime with metronidazole, and ampicillin-sulbactam to which only 21.6% of bacteria were susceptible. Bacterial growths and higher resistances correlate to an elevated risk of complications. Rare bacteria are found in many patients, but there is no specific consequence regarding antibiotic susceptibility, clinical course, or complications. Prospective, comprehensive studies are needed to further elicit pediatric appendicitis microbiology and antibiotic treatment.

Ceftazidime/avibactam-resistant meropenem-susceptible KPC-producing Klebsiella pneumoniae: Analysis of cases and evaluation of in vitro activity of fosfomycin-containing combinations.

OBJECTIVES: Little is known regarding outcomes and optimal therapeutic regimens of infections caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) resistant to ceftazidime/avibactam (CZA) and susceptible to meropenem (MEM). Although susceptible to MEM in vitro, the possibility of developing MEM resistance overtime is a concern. We describe the clinical characteristics of patients with colonization/infection due to KPC variants with a focus on the in vitro activity of fosfomycin (FOS)-containing combinations. METHODS: Patients with colonization/infection due to a KPC variant were included. Fosfomycin susceptibility was performed by agar dilution method. Synergistic activity of FOS-based combinations was evaluated by gradient strip-agar diffusion method. The emergence of in vitro MEM resistance was also tested. RESULTS: Eleven patients were included: eight with infection [four with ventilator-associated pneumonia and four with bloodstream infections] and three with colonization. Previous therapy with CZA was administered to all patients (with a mean cumulative duration of 23 days). All subjects with infection received meropenem, in monotherapy (n = 4) or with amikacin (n = 2) or fosfomycin (n = 2), and achieved clinical cure. A new CZA-susceptible and MEM-resistant KPC-Kp strain was subsequently isolated in three patients (27.3%). Meropenem/vaborbactam (MVB) showed high in vitro activity, while FOS+MEM combination was synergistic in 40% of cases. In vitro resistance to MEM was observed with maintenance of CZA resistance. CONCLUSIONS: Detection of KPC variants may occur within the same patient, especially if CZA has been previously administered. Although clinical success has been obtained with carbapenems, the emergence of MEM resistance is a concern. Fosfomycin plus meropenem is synergistic and may be a valuable combination option for KPC variants, while MVB may be considered in monotherapy. The detection of KPC variants in an endemic setting for KPC-Kp represents a worryingly emerging condition. The optimal therapeutic approach is still unknown and the development of meropenem resistance is of concern, which may lead to therapeutic failure in clinical practice. In these cases, the addition of fosfomycin to meropenem, or a more potent antibiotic, such as meropenem/vaborbactam, may be valuable therapeutic options.

Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico.

BACKGROUND: Leclercia adecarboxylata is a bacteria closely related to Escherichia coli according to its biochemical characteristics and is commonly considered non-pathogenic although a growing number of publications classify it as an emerging pathogen. Fosfomycin resistance is a common trait for L. adecarboxylata encoded by fosALA gene. OBJECTIVE: To analyze genomic traits of sixteen L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition. METHODS: Twenty-eight L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition were identified biochemically with a Vitek ® automated system. The strains were phenotyped by their growth on Eosin Methylene Blue agar or MacConkey agar plates. Additionally, Pulsed field gel electrophoresis (PFGE) was performed to establish the clonal relationship. The genomic DNA of sixteen strains was obtained using a Qubit ® dsDNA HS Assay Kit and sequenced on an Illumina ® MiSeq instrument. Draft genomes were assembled using PROKKA and Rast. Assemblies were submitted to Resfinder and PathogenFinder from the Center for Genomic Epidemiology in order to find resistance genes and pathogenic potential. IslandViewer4 was also used to find Pathogenicity and Phage Islands. For identification of the fosA gene, manual curation and Clustal analysis was performed. A novel FosA variant was identified. Finally, phylogenetic analysis was performed using VAMPhyRE software and Mega X. RESULTS: In this paper, we report the genomes of sixteen strains of Leclercia adecarboxylata causing an outbreak associated with parenteral nutrition in public hospitals in Mexico. The genomes were analyzed for genetic determinants of virulence and resistance. A high pathogenic potential (pathogenicity index 0.82) as well as multiple resistance genes including carbapenemics, colistin and efflux pumps were determined. Based on sequence analysis, a new variant of the fosALA gene was described. Finally, the outbreak was confirmed by establishing the clonal relationship among the sixteen genomes obtained. CONCLUSIONS: Commensal strains of L. adecarboxylata may acquire genetic determinants that provide mechanisms of host damage and go unnoticed in clinical diagnosis. L. adecarboxylata can evolve in a variety of ways including the acquisition of resistance and virulence genes representing a therapeutic challenge in patient care.

Australian Group on Antimicrobial Resistance (AGAR) Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP).

Abstract: From 1 January to 31 December 2021, forty-eight institutions around Australia participated in the Australian Staphylococcus aureus Surveillance Outcome Programme (ASSOP). The aim of ASSOP 2021 was to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that were antimicrobial resistant, with particular emphasis on susceptibility to methicillin and on characterisation of the molecular epidemiology of the methicillin-resistant isolates. A total of 2,928 SAB episodes were reported, of which 78.4% were community-onset. Overall, 16.9% of S. aureus isolates were methicillin resistant. The 30-day all-cause mortality associated with methicillin-resistant SAB was 15.0%, which was not significantly different from the 14.4% all-cause mortality associated with methicillin-susceptible SAB (p = 0.7). With the exception of the ß-lactams and erythromycin, antimicrobial resistance in methicillin-susceptible S. aureus was rare. However, in addition to the ß-lactams, approximately 36% of methicillin-resistant S. aureus (MRSA) were resistant to ciprofloxacin; 30% to erythromycin; 15% to tetracycline; 16% to gentamicin; and 3% to co-trimoxazole. When applying the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, teicoplanin resistance was detected in three S. aureus isolates. Resistance to vancomycin or linezolid was not detected. Resistance to non-ß-lactam antimicrobials was largely attributable to the healthcare-associated MRSA (HA-MRSA) clone ST22-IV [2B] (EMRSA-15), and the community-associated MRSA (CA-MRSA) clone ST45-V [5C2&5] which has acquired multiple antimicrobial resistance determinants including ciprofloxacin, erythromycin, clindamycin, gentamicin and tetracycline. The ST22-IV [2B] (EMRSA-15) clone is the predominant HA-MRSA clone in Australia. Nonetheless, 85% of methicillin-resistant SAB episodes were due to CA-MRSA clones. Although polyclonal, approximately 68% of CA-MRSA clones were characterised as ST93-IV [2B] (Queensland clone); ST45-V [5C2&5]; ST5-IV [2B]; ST1-IV [2B]; ST30-IV [2B]; and ST97-IV [2B]. As CA-MRSA is well established in the Australian community, it is important to monitor antimicrobial resistance patterns in community- and healthcare-associated SAB as this information will guide therapeutic practices in treating S. aureus bacteraemia.

INVESTIGATION OF TIGECYCLINE SUSCEPTIBILITY OF MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII ISOLATES BY DISC DIFFUSION, AGAR GRADIENT AND BROTH MICRODILUTION TESTS.

The use of tigecycline is becoming increasingly important because of the high levels of antibiotic resistance in Acinetobacter baumannii (A. baumannii) isolates. In this prospective study, multidrug-resistant A. baumannii isolates were obtained from various tissue and fluid samples of patients admitted to or treated at various departments and tested in Laboratory of Microbiology, Duzce University Medical Faculty between January 2013 and December 2015. Tigecycline resistance in multidrug-resistant A. baumannii isolates were analyzed using the disc diffusion test (DDT), agar gradient test (AGT), and gold standard test [broth microdilution test (BMT)]. A. baumannii isolates resistant to multiple drugs were included in the study (N=94). Using the BMT method, 89 (95%), 4 (4%) and 1 (1%) A. baumannii isolates were determined as tigecycline susceptible, intermediate and resistant isolates, respectively. Using the Food and Drug Administration criteria, the rates of major error (ME), minor error (mE) and categorical agreement (CA) for DDT were 26%, 67% and 9%, respectively. In contrast, for AGT, the rates of ME, mE and CA were 0%, 4%, 95%, respectively. Tigecycline resistance as assessed by BMT showed no increase between 2013 and 2015. Accordingly, isolates found to be resistant or intermediate by DDT should be confirmed by BMT. Due to the ease of application, AGT is a safe method of detecting susceptibility.

Virulence, phenotype and genotype characteristics of invasive group B Streptococcus isolates obtained from Swedish pregnant women and neonates.

Group B streptococci (GBS) are bacteria that can cause preterm birth and invasive neonatal disease. Heterogeneous expression of virulence factors enables GBS to exist as both commensal bacteria and to become highly invasive. A molecular epidemiological study comparing GBS bacterial traits, genotype and host characteristics may indicate whether it is possible to predict the risk of perinatal invasive GBS disease and more accurately target intrapartum antibiotic prophylaxis. A total of 229 invasive GBS isolates from Swedish pregnant women or neonates were assessed for virulence and phenotypic traits: hemolysis zone, hemolytic pigment (Granada agar), Streptococcus B Carrot Broth (SBCB) assay, CAMP factor, and hyaluronidase activity. Genes regulating hemolytic pigment synthesis (covR/covS, abx1, stk1, stp1) were sequenced. Of the virulence factors and phenotypes assessed, a Granada pigment or SBCB score ≥ 2 captured more than 90% of EOD isolates with excellent inter-rater reliability. High enzyme activity of hyaluronidase was observed in 16% (36/229) of the invasive GBS isolates and notably, in one case of stillbirth. Hyaluronidase activity was also significantly higher in GBS isolates obtained from pregnant/postpartum individuals versus the stillbirth or neonatal invasive isolates (p < 0.001). Sequencing analysis found that abx1 (g.T106I), stk1 (g.T211N), stp1 (g.K469R) and covS (g.V343M) variants were present significantly more often in the higher (Granada pigment score ≥ 2) versus lower pigmented isolates (p < 0.001, each variant). Among the 203 higher Granada pigment scoring isolates, 22 (10.8%) isolates had 3 of the four sequence variants and 10 (4.9%) had 2 of the four sequence variants. Although heterogeneity in GBS virulence factor expression was observed, the vast majority were more highly pigmented and contained several common sequence variants in genes regulating pigment synthesis. High activity of hyaluronidase may increase risk for stillbirth and invasive disease in pregnant or postpartum individuals. Our findings suggest that testing for GBS pigmentation and hyaluronidase may, albeit imperfectly, identify pregnant people at risk for invasive disease and represent a step towards a personalized medical approach for the administration of intrapartum antibiotic prophylaxis.

High-frequency devices effect in vitro: promissing approach in the treatment of acne vulgaris?

BACKGROUND: Acne vulgaris is an inflammatory skin disorder leading to an impairment of quality of life and is therefore not only a cosmetic issue. Its pathogenesis is multifactorial - of particular importance is the colonization with the bacterium Propionibacterium acnes. A wide range of different treatment options exists including topical and systemic treatments depending on severity. High Frequency (HF) therapy, historically developed in the 19th century, claims antimicrobial effects on acne skin, but solid data on its efficacy and mechanism of action is lacking. OBJECTIVES: The main objective of this study was to determine the efficacy of HF therapy on skin flora and P. acnes in vitro using a commercial device as well as to review studies on the mechanism of action. METHODS: The plasma source was investigated regarding electrical settings, heat, and ozone development. Bacterial skin flora, fungal isolates, and P. acnes were exposed to HF in vitro and compared to unexposed controls by evaluating the number of colonies on agar plates. To further analyze bacterial species from normal skin flora, 16S-sequencing was performed. Statistical analyses were carried out using row analysis and unpaired t-test. RESULTS: HF treatment led to a significant reduction of almost every bacterial and fungal species investigated in this study. Moreover, the number of colonies forming units was significantly decreased in P. acnes after HF treatment compared to controls in vitro. STUDY LIMITATIONS: The experiments were performed in vitro only. To assess clinical effects further in vivo experiments are necessary. CONCLUSIONS: The results collected in this study, although in vitro, provide a mechanistic basis for HF as a complementary treatment option for patients with acne. It might also have a beneficial effect on patients with superficial infectious skin of the skin.

Detection of differentially culturable tubercle bacteria in sputum from drug-resistant tuberculosis patients.

Several studies described the presence of non-replicating, drug-tolerant differentially culturable tubercle bacteria (DCTB) in sputum from patients with active tuberculosis (TB). These organisms are unable to form colonies on agar but can be recovered in liquid media supplemented with culture filtrate as a source of growth factors. Herein, we undertook to investigate the response of DCTB during the treatment of individuals with drug-resistant TB. A cohort of 100 participants diagnosed with rifampicin-resistant TB were enrolled and prospectively followed to monitor response to therapy using routine culture and limiting dilution assays, supplemented with culture filtrate (CF) to quantify DCTB. Fifteen participants were excluded due to contamination, and of the remaining 85 participants, 29, 49, and 7 were infected with rifampicin mono-resistant (RMR), multidrug-resistant (MDR), or extremely drug-resistant (XDR) TB, respectively. Analysis of baseline sputum demonstrated that CF supplementation of limiting dilution assays detected notable amounts of DCTB. Prevalence of DCTB was not influenced by smear status or mycobacterial growth indicator tube time to positivity. CF devoid of resuscitation promoting factors (Rpfs) yielded a greater amount of DCTB in sputum from participants with MDR-TB compared with those with RMR-TB. A similar effect was noted in DCTB assays without CF supplementation, suggesting that CF is dispensable for the detection of DCTB from drug-resistant strains. The HIV status of participants, and CD4 count, did not affect the amount of DCTB recovered. During treatment with second-line drug regimens, the probability of detecting DCTB from sputum specimens in liquid media with or without CF was higher compared with colony forming units, with DCTB detected up to 16 weeks post treatment. Collectively, these data point to differences in the ability of drug-resistant strains to respond to CF and Rpfs. Our findings demonstrate the possible utility of DCTB assays to diagnose and monitor treatment response for drug-resistant TB, particularly in immune compromised individuals with low CD4 counts.

[The Role of Emergency Departments in Cross-Contamination: The Case of VRE]. / Çapraz Bulasta Acil Servislerin Rolü: VRE Örnegi.

Colonized surfaces, equipment, individuals, and infected patients can be sources for the hospital spread of vancomycin-resistant enterococci (VRE), which is one of the important nosocomial pathogens. The basic epidemiological tools for the prevention and control of hospital-acquired infections are the typing methods. Pulsed-field gel electrophoresis (PFGE) is a highly discriminating method used frequently to detect clonal associations in epidemics and hospital-acquired VRE infections. This study aimed to investigate the presence of cross-contamination, which service or services come to the forefront in case of possible cross-contamination and clonal relationship between VRE strains isolated from rectal swab, clinical and environmental swab samples taken in two different periods in various clinics of Istanbul University Istanbul Medical Faculty Hospital by PFGE method. A total of 125 VREs isolated in two different periods were included in the study. Rectal and environmental swab samples were inoculated on Enterococcosel agar and sodium azide broth, urine samples were inoculated on chromogenic agar, and other clinical samples were inoculated on 5% sheep blood agar and incubated for 18-24 hours. VITEK 2 Compact automation system GP panel (bioMerieux, MarcyL'Etoile, France) was used for the species identification of catalase-negative, gram-positive colonies and disc diffusion and minimum inhibitory concentration (MIC) gradient tests were used to determine vancomycin susceptibility. Multiplex polymerase chain reaction was used to search for vanA and vanB resistance genes in isolates identified as VRE, and PFGE was used to determine clonal association. All isolates were identified as Enterococcus faecium with the vanA resistance gene. It was shown that PFGE clones were divided into six types with 65% similarity (A-F), and in this polyclonal spread, the major type was type A, which contained 108 isolates in 37 subtypes existed in the hospital for years. In patients from whom similar isolates were obtained, the high rate of hospitalizations in the same emergency room or in different emergency services in the same building drew attention. Our results showed that the presence of VRE was established in our hospital, new isolates were added from time to time, and there was a cross-contamination. It was observed that emergency services, where infection control measures were neglected due to working conditions, were among the high-risk areas for VRE contamination. In order to better understand the importance of emergency services in cross-contamination, it would be useful to conduct surveillance studies among patients hospitalized in emergency services and monitor the rate of VRE in the services where positive patients were transferred.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

Vaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 µg/mL) as well as minimum fungicidal concentrations (2 to 32 µg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 µg/mL and 32 µg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis. IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Identification of Mutations Associated With Macozinone-Resistant in Mycobacterium Tuberculosis.

Novel anti-tuberculosis drug macozinone (MCZ) is identified as a drug candidate and is currently under clinical development for the treatment of tuberculosis. However, the mutations conferring resistance to MCZ remain inadequately characterized. In this study we investigated resistant mutations to the MCZ through de novo resistance selection in vitro. This was accomplished by passing the Mycobacterium tuberculosis H37Rv on solid agar plates, which were infused with the antibiotic. Through whole genome sequencing and targeted PCR genomic resistance conferring SNPs were catalogued, and the resistant phenotype was analyzed by MABA test. Of MCZ-resistant clones obtained in vitro the mutation of C387S in dprE1 is only observed in high-level resistant clones (MIC90 > 500 ng/mL) indicating that C387S mutation is directly related to high-level MCZ-resistance. In addition, high-level resistance to MCZ can occur in clone grew on agar plates infused with low concentration MCZ, which means low concentration MCZ induction can produce high-level drug-resistance and it is easy to produce high-level MCZ-resistance. We also found two new mutations (G61A and G248A) in dprE1 for the first time. The MIC90 of other clones except the clones carrying the C387S mutation was at the same level (20 ng/mL > MIC90 > 2 ng/mL). Of low-level resistant clones other gene mutations involved in drug efflux or membrane permeability were found (pepQ, Rv0678, arsC, etc.), with highest mutation frequency in Rv0678 (50/64, 78.12%). It suggests that there may be new mechanisms independent of dprE1 mutations.

Toxic non-inflammatory fungal keratitis.

Purpose: To report a new entity called "toxic non-inflammatory fungal keratitis." Methods: Eyes manifesting infective keratitis with a history of prior administration of topical steroids were included in the study. The details pertaining to the type of injury, duration of injury, and primary treatment for corneal trauma were meticulously documented. The corneal tissues were scraped from the patients and were analyzed for fungal filaments by using a 10% KOH mount under a compound microscope. Moreover, these scraped materials were plated on blood agar and Sabouraud dextrose agar plates. Results: The corneal ulcers displayed a disproportionately reduced intensity of pain and improved visual acuity. Further, 10% KOH revealed profuse fungal filaments with few inflammatory cells in all the patients. The anterior chamber cells and flare were either reduced or entirely absent. There was no evidence of lid edema and surrounding corneal edema in any of the patients. The mean healing period was 28.8 days (standard deviation (SD): 10.05). The KOH mount revealed the presence of confluent fungal hyphae with a few inflammatory cell infiltrates. The Aspergillus species and Fusarium species were found in 47% and 40% of the cases, respectively. Conclusion: Toxic non-inflammatory fungal keratitis following steroid therapy needs to be considered in fungal ulcers with disproportionately less pain and good visual acuity. The fungal ulcers with altered clinical signs of classical inflammation need to be assessed for topical steroid misuse.

Periprosthetic knee joint infection caused by Sneathia sanguinegens.

Sneathia sanguinegens is a fastidious, Gram-negative, rod-shape organism rarely isolated from human specimens. In the present report, we describe a case of periprosthetic knee joint infection due to the organism, which occurred in a female patient receiving immunosuppressants for underlying lupus nephritis. The causative organism was isolated from the synovial fluid in the affected knee joint through inoculating the material on chocolate agar and incubation for 15 days under 5% CO2. Moreover, the organism was capable to be subcultured on chocolate agar with incubation for a few days under 5% CO2, demonstrating that this uncommon organism, although generally considered as a strict anaerobe, is culturable in aerobic condition if appropriate media and a sufficient incubation time are given. The patient was treated with intravenous cefepime, an antibiotic highly active to the isolated organism in an in vitro study, in addition to intraarticular debridement and exchanging a polyethylene insert in the affected joint. The antimicrobial therapy with cefepime was given for 19 days and, thereafter, changed with oral levofloxacin. Although the patient showed full recovery after administration of levofloxacin for 100 days, an in vitro study conducted later revealed that levofloxacin was inactive to the isolated organism.

Australian Group on Antimicrobial Resistance (AGAR) Australian Staphylococcus aureus Sepsis Outcome Programme (ASSOP) Annual Report 2020.

From 1 January to 31 December 2020, forty-nine institutions around Australia participated in the Australian Staphylococcus aureus Sepsis Outcome Programme (ASSOP). The aims of ASSOP 2020 were to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that were antimicrobial resistant, with particular emphasis on susceptibility to methicillin; and to characterise the molecular epidemiology of the methicillin-resistant isolates. A total of 2,734 SAB episodes were reported, of which 79.7% were community-onset. Of S. aureus isolates, 17.6% were methicillin resistant. The 30-day all-cause mortality associated with methicillin-resistant SAB was 14.2%, which was not significantly different from the 13.3% mortality associated with methicillin-susceptible SAB (p = 0.6). With the exception of the ß-lactams and erythromycin, antimicrobial resistance in methicillin-susceptible S. aureus was rare. However, in addition to the ß-lactams, approximately 35% of methicillin-resistant S. aureus (MRSA) were resistant to erythromycin, 33% to ciprofloxacin, 13% to tetracycline, 13% to gentamicin and 4% to co-trimoxazole. When applying the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, teicoplanin resistance was detected in four S. aureus isolates. Resistance was not detected for vancomycin and linezolid. Resistance to non-beta-lactam antimicrobials was largely attributable to two healthcare-associated MRSA (HA-MRSA) clones: ST22-IV [2B] (EMRSA-15) and ST239-III [3A] (Aus-2/3 EMRSA). The ST22-IV [2B] (EMRSA-15) clone is the predominant HA-MRSA clone in Australia. However, 85% percent of methicillin-resistant SAB isolates were community-associated MRSA (CA-MRSA) clones. Although polyclonal, approximately 77% of CA-MRSA clones were characterised as: ST93-IV [2B] (Queensland CA-MRSA); ST5-IV [2B]; ST45-V [5C2&5]; ST1-IV [2B]; ST30-IV [2B]; ST8-IV [2B]; and ST97-IV [2B]. The CA-MRSA clones, in particular ST45-V [5C2&5], have acquired multiple antimicrobial resistance determinants including ciprofloxacin, erythromycin, clindamycin, gentamicin and tetracycline. The multi-resistant ST45-V [5C2&5] clone accounted for 11.0% of CA-MRSA. As CA-MRSA is well established in the Australian community, it is important to monitor antimicrobial resistance patterns in community- and healthcare-associated SAB as this information will guide therapeutic practices in treating S. aureus sepsis.

Implications and Efficacy of Aromatase Inhibitors in Combination and Monotherapy for the Treatment of Lung Cancer.

BACKGROUND: Lung tumors express high levels of aromatase enzyme compared to surrounding normal tissue. Inhibition of aromatase has emerged as a recent therapeutic approach for the treatment of breast cancer. However, the role of aromatase inhibition in lung cancer treatment requires further investigation. METHODS: The anti-proliferative effects of aromatase inhibitors were evaluated by MTT assay. Cell migration was assessed using a wound healing assay. The mechanism of cell death was determined using the annexin VFITC/ propidium iodide staining flow cytometry method. The soft agar colony formation assay evaluated cells' capability to form colonies. RESULT: Exemestane and curcumin significantly inhibited the growth of lung cancer cell lines in a dose- and timedependent manner. The IC50 values after 48 hours of treatment with exemestane were 176, 180, and 120 µM in A549, H661, and H1299, respectively. Curcumin IC50 values after 48 hours were 80, 43, and 68 µM in A549, H661, and H1299, respectively. The combined treatment of exemestane or curcumin with cisplatin, raloxifene, and celecoxib resulted in a synergistic effect in the A549 lung cell line with a combination index of less than 1, suggesting synergism. Exemestane resulted in approximately 96% inhibition of wound closure at 100 µM, while curcumin resulted in approximately 63% inhibition of wound closure at 50 µM. Exemestane and curcumin inhibited the formation of cell colonies by reducing the number and size of formed colonies of A549, H661, and H1299 cell lines in a concentration dependent manner. Exemestane and curcumin had significantly induced apoptosis in A549 cells compared to control of untreated cells. CONCLUSION: Aromatase inhibition by exemestane or curcumin had significantly inhibited the growth of lung cancer cell lines, synergized with cisplatin, raloxifene, and celecoxib, suppressed lung cancer cell migratory potential, induced apoptosis, and reduced colony formation of lung cancer cells.

Pythium insidiosum keratitis - A review.

Pythium insidiosum is an oomycete and is also called "parafungus" as it closely mimics fungal keratitis. The last decade saw an unprecedented surge in Pythium keratitis cases, especially from Asia and India, probably due to growing research on the microorganism and improved diagnostic and treatment modalities. The clinical features such as subepithelial infiltrate, cotton wool-like fluffy stromal infiltrate, satellite lesions, corneal perforation, endoexudates, and anterior chamber hypopyon closely resemble fungus. The classical clinical features of Pythium that distinguish it from other microorganisms are reticular dots, tentacular projections, peripheral furrowing, and early limbal spread, which require a high index of clinical suspicion. Pythium also exhibits morphological and microbiological resemblance to fungus on routine smearing, revealing perpendicular or obtuse septate or aseptate branching hyphae. Culture on blood agar or any other nutritional agar is the gold standard for diagnosis. It grows as cream-colored white colonies with zoospores formation, further confirmed using the leaf incarnation method. Due to limited laboratory diagnostic modalities and delayed growth on culture, there was a recent shift toward various molecular diagnostic modalities such as polymerase chain reaction, confocal microscopy, ELISA, and immunodiffusion. As corneal scraping (10% KOH, Gram) reveals fungal hyphae, antifungals are started before the culture results are available. Recent in vitro molecular studies have suggested antibacterials as the first-line drugs in the form of 0.2% linezolid and 1% azithromycin. Early therapeutic keratoplasty is warranted in nonresolving cases. This review aims to describe the epidemiology, clinical features, laboratory and molecular diagnosis, and treatment of Pythium insidiosum keratitis.