Bloodstream Isolates Research Articles

Clonal outbreak of Candida vulturna in a paediatric oncology ward in Maranhão, Brazil

ObjectiveTo describe an outbreak due to Candida vulturna, a newly emerging Candida species belonging to the Candida haemulonii species complex in the Metschnikowiaceae family. MethodsIn this retrospective cohort study we genotyped 14C. vulturna bloodstream isolates, occurring in a 4-month-period in paediatric cancer patients in a Brazilian hospital. To prove an outbreak, ITS sequence analysis and whole genome sequencing (WGS) was done. Antifungal susceptibility was performed with the reference CLSI method and the commercial Sensititre YeastOne (SYO) YO10 plates. A control C. vulturna isolate from another region in Brazil was included in all analyses. ResultsMALDI-TOF-MS identified isolates as C. pseudohaemulonii and C. duobushaemulonii albeit with low scores and therefore molecular methods were required for accurate identification. ITS sequence analyses clearly differentiated C. vulturna from other species in the C. haemulonii species complex. WGS proved the presence of a clonal outbreak with C. vulturna involving 14 paediatric patients. Antifungal susceptibility testing (AFST) with two methods showed the isolates had low MICs of commonly available antifungals. ConclusionThis study describes an outbreak due to the rare yeast C. vulturna, related to C. auris, during a four-month period in patients admitted to a paediatric oncology ward in a Brazilian hospital. In contrast to previous studies the yeast was susceptible to all antifungals and patient outcome was good.

High Mortality Rates and Antimicrobial Resistance in Children with Invasive Bacterial Infections in a Tertiary Care Facility in West Africa

Abstract Background Childhood data on infection mortality and antimicrobial resistance in Sub-Saharan Africa are emerging but remain limited. The University of Maryland Center for Vaccine Development (CVD) and CVD-Mali have been conducting a prospective observational study at L’Hopital Gabriel Toure (HGT), a referral hospital in Bamako, Mali, since 2002 to identify causes of invasive bacterial infections (IBI) in children. These data have been used to initiate vaccination programs to decrease the burden of Haemophilus influenzae type b and Streptococcus pneumoniae. Additional demographic and antimicrobial susceptibility data have been collected since 2021, allowing for more detailed resistance and mortality analyses. Methods All children presenting to HGT are screened. Children ages 0-15 years with temperature of at least 39 degrees Celsius and/or suspicion of IBI are eligible. Eligible children have blood cultures collected with additional site-specific cultures when focal infection is suspected. Cultures are performed using the Bactec system; when positive, Gram staining, sub-culturing, and antimicrobial susceptibility testing (by disk diffusion using standard methods) are performed. Organisms thought to be contaminants are excluded. American type culture collection strains are utilized for quality control. Results Between 1/14/2021 and 9/14/2023, 28703 patients were screened. 2156 patients were included. 212 patients (10%) had 235 pathogens isolated. Gram positive organisms were most frequently isolated (N=112), followed by Gram negative Enterobacterales (N=81), non-Enterobacterales Gram negatives (N=36), and yeast (N=6). Staphylococcus aureus was the most frequently isolated Gram positive organism (59 bloodstream isolates, 2 joint isolates, 2 subcutaneous isolates) followed by S.pneumoniae (10 bloodstream isolates, 17 cerebrospinal fluid isolates, 1 joint isolate) and Enterococcus species (16 bloodstream isolates). Klebsiella pneumoniae (28 bloodstream isolates, 2 cerebrospinal fluid isolates) and Escherichia coli (18 bloodstream isolates, 3 cerebrospinal fluid isolates, 2 subcutaneous isolates) were the most frequently isolated Gram negative Enterobacterales, with Pseudomonas species most common among non-Enterobacterales Gram negative organisms (16 bloodstream isolates, 1 cerebrospinal fluid isolate). Mortality rates in both culture-positive and culture-negative patients were high (52% and 28%, respectively), with greater than 50% of deaths occurring within 48 hours in both groups. 88% of Gram negative Enterobacterales were ceftriaxone resistant or intermediate, with 56% ciprofloxacin resistant or intermediate; 55% of isolates were resistant or intermediate to both. 71% of isolates were gentamicin resistant. Imipenem resistance remained low, with 91% of isolates retaining sensitivity. 24% of S. aureus isolates were oxacillin resistant and 34% of isolates were resistant or intermediate to trimethoprim/sulfamethoxazole. 100% of S. aureus isolates tested (14/60) retained sensitivity to clindamycin. Only 54% of pneumococcal isolates had positive oxacillin screens, implying frequent penicillin resistance, and only 60% of enterococcal species were ampicillin sensitive, although 92% retained vancomycin sensitivity. Conclusion We found higher mortality rates in our study than in similar studies; the contribution of issues such as limited access to advanced supportive care and delay in presentation is an area for further study. The high frequency of multidrug-resistant organisms is alarming, suggesting an urgent need to investigate underlying contributing factors, increase access to affordable broad-spectrum antibiotics, and create robust antimicrobial stewardship programs.

Characteristics of Oral Acinetobacter spp. and Evolution of Plasmid-Mediated Carbapenem Resistance in Bacteremia Patients with Hematological Malignancies.

Patients with hematological malignancies are more susceptible to infections, leading to a poor prognosis. Acinetobacter colonization is a risk factor for secondary bacteremia. Antibiotic susceptibility phenotypes and genomic characteristics of 48 oral Acinetobacter spp. and one bloodstream Acinetobacter baumannii from patients with hematological malignancies were analyzed by antimicrobial susceptibility tests and whole-genome sequencing. We conducted comparative genomic analysis of oral and blood isolates from the same patient. A. baumannii was the most common (72.92%, 35/48) Acinetobacter species in oral Acinetobacter spp. isolates. Seventeen different A. baumannii sequence types were identified using the Pasteur MLST scheme; however, the dominant global clones GC1 and GC2 were not present. Among the isolates, 46 (95.8%) were carbapenem-susceptible Acinetobacter spp. One patient treated with meropenem for 15 days developed A. baumannii bacteremia 46 days after the isolation of oral A. baumannii AOR07. Oral and bloodstream isolates from the same patient were closely related to only four non-synonymous mutations on the chromosome. The bla OXA-58 gene was transferred between plasmids through XerCD-mediated recombination, leading to an elevated copy number, causing carbapenem resistance in bloodstream isolates. Oral Acinetobacter spp. may cause secondary bacteremia. The amplification and transfer of bla OXA-58 in the plasmids explained the increased carbapenem resistance in the bloodstream isolate.

Antifungal Susceptibility Data and Epidemiological Distribution of Candida spp.: An In Vitro Five-Year Evaluation at University Hospital Policlinico of Catania and a Comprehensive Literature Review.

Invasive fungal infections represent a concerning healthcare issue, with Candida spp. reported as the main aetiological agent. Candida spp. bloodstream infections show high mortality rates, indicating increasing antifungal-resistance episodes as a contributing feature. Despite the global prevalence of C. albicans, non-albicans species emerged as significant in the last decades. The present manuscript reports a five-year evaluation on Candida spp. bloodstream isolates and their antifungal susceptibility profiles, aiming to enrich the literature and epidemiological data. According to the gathered data, antifungal-resistance cases remained uncommon. However, the study revealed rare resistance phenotypes such as a single case of pan-echinocandin resistance C. albicans. Finally, a comprehensive review of Candida spp. antifungal resistance integrates the data, emphasizing the extreme species-specific variability and the consequent importance of always providing species identification.

Exploring the Arsenal of Novel Antifungal Drug Targets for Combating Fungal Infections.

Fungal infections contribute to over 1.5 million fatalities each year, with cutaneous mycoses standing as prominent global infections. The spectrum of these mycoses varies widely, encompassing enduring afflictions like ringworm, localized infections such as tinea capitis, recurrent instances like vaginal candidiasis, and potentially fatal systemic infections impacting multiple organ systems. The escalating recognition of the health and socioeconomic ramifications associated with fungal pathogens underscores their importance in contemporary discourse. On a global scale, projections indicate that over 300 million individuals experience significant fungal infections annually, resulting in a mortality rate exceeding 1.5 million deaths per year. Alarmingly, resistance to commonly used antifungal drugs was on the rise, with some reports suggesting that over 10% of Candida bloodstream isolates worldwide were resistant to fluconazole, a commonly prescribed antifungal medication. Therefore, there is an immediate need to increase the accessibility of new antifungal medications while minimizing their costs and adverse effects. Fungi, as heterotrophic organisms, acquire nutrients through absorption. Their filamentous structure, composed of hyphae, facilitates efficient nutrient uptake by secreting enzymes that break down complex organic matter into simpler compounds. These organisms exhibit remarkable adaptability in responding to environmental cues, adjusting growth rates, and altering morphological features. Fungi regulate their metabolism intricately, undergoing various metabolic pathways for energy production and utilizing diverse substrates for respiration. Additionally, they exhibit distinctive reproductive strategies, employing both sexual and asexual modes of reproduction, contributing to their genetic diversity and resilience in diverse ecosystems. We now have more information than ever on the origins of infection as well as the physiology of fungi cells, giving us the chance to use it to produce new generations of antifungals. This review includes various novel antifungal drug targets showing their possible effects via different mechanisms aiming at vital functions like GPI synthesis, cell wall synthesis, hyphal growth, and other essential pathways responsible for fungal growth.

Unraveling novel mutation patterns and morphological variations in two dalbavancin-resistant MRSA strains in Austria using whole genome sequencing and transmission electron microscopy

BackgroundThe increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to non-beta-lactam antimicrobials poses a significant challenge in treating severe MRSA bloodstream infections. This study explores resistance development and mechanisms in MRSA isolates, especially after the first dalbavancin-resistant MRSA strain in our hospital in 2016.MethodsThis study investigated 55 MRSA bloodstream isolates (02/2015–02/2021) from the University Hospital of the Medical University of Vienna, Austria. The MICs of dalbavancin, linezolid, and daptomycin were assessed. Two isolates (16–33 and 19–362) resistant to dalbavancin were analyzed via whole-genome sequencing, with morphology evaluated using transmission electron microscopy (TEM).ResultsS.aureus BSI strain 19–362 had two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene. Isolate 16–33 had a 534 bp deletion in the DHH domain of GdpP and a SNV in pbp2 (p.G146R). Both strains had mutations in the rpoB gene, but at different positions. TEM revealed significantly thicker cell walls in 16–33 (p < 0.05) compared to 19–362 and dalbavancin-susceptible strains. None of the MRSA isolates showed resistance to linezolid or daptomycin.ConclusionIn light of increasing vancomycin resistance reports, continuous surveillance is essential to comprehend the molecular mechanisms of resistance in alternative MRSA treatment options. In this work, two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene were newly identified as possible causes of dalbavancin resistance.

Antibiotics stimulates the development of persistent cells in biofilms of Candida albicans bloodstream isolates

Candida albicans invasive candidiasis is considered a global health problem. In such cases, biofilm formation on implanted devices represents a therapeutic challenge and the presence of metabolically inactive persistent cells (PCs) in these communities increases their tolerance to fungicidal drugs. This study investigated the influence of amoxicillin, AMX; cefepime, CEF; gentamicin, GEN; amikacin, AMK; vancomycin, VAN; and ciprofloxacin, CIP; on the production of PCs in biofilms of C. albicans bloodstream isolates. 48 h-mature biofilms (n = 6) grown in RPMI-1640 supplemented with antibiotics were treated with 100 μg ml−1 amphotericin B and then evaluated for PCs. Biofilms grown in the presence of antibiotics produced more PCs, up to 10×, when exposed to AMX and CIP; 5 × to CEF; and 6 × to GEN and VAN. The results indicate that antibiotics can modulate PC production in C. albicans biofilms. This scenario may have clinical repercussions in immunocompromised patients under broad-spectrum antibiotic therapy.

Nosocomial transmission of fluconazole-resistant Candida glabrata bloodstream isolates revealed by whole-genome sequencing.

The prevalence of fluconazole-resistant bloodstream infections caused by Candida glabrata is increasing globally, but the transmission of these resistant strains within hospitals has rarely been documented. Through whole-genome sequencing and epidemiological analyses, this study identified two potential clusters of C. glabrata bloodstream infections within the same hospital, revealing the transmission of clonal C. glabrata strains with different levels of fluconazole susceptibility or distinct transcription factor pleiotropic drug resistance protein 1 (Pdr1p) single-nucleotide polymorphism profiles among patients receiving antifungal therapy.

Comparative in vitro efficacy of antibiotics against the intracellular reservoir of Staphylococcus aureus.

We assessed antimicrobial activity at phagolysosome-mimicking pH, intracellular penetration, and SA eradication within KCs in vitro for clinically prescribed antistaphylococcal agents alone or in combination: vancomycin, daptomycin, ceftaroline, ceftobiprole, oritavancin, oxacillin, cefazolin; rifampin and fosfomycin. pH-adjusted broth microdilution assays, intracellular bioaccumulation assays, and intracellular killing assays against clinical bloodstream isolates were performed using a murine KC line with study agents. Rifampin and β-lactams exhibited enhanced activity [2- to 16-fold minimum inhibitory concentrations (MIC) decrease] at phagolysosomal pH while vancomycin, oritavancin, daptomycin and fosfomycin demonstrated reduced activity (2- to 32-fold MIC increase in order of least to greatest potency reduction). All agents evaluated had poor to modest intracellular to extracellular concentration ratios (0.024-7.8), with exceptions of rifampin and oritavancin (intracellular to extracellular ratios of 17.4 and 78.2, respectively). Finally, we showed that the first-line treatment for SA bacteraemia (SAB), vancomycin, performed worse than all other tested antibiotics in eradicating intracellular SA at human Cmax concentration (0.20 log cfu decrease), while oritavancin performed better than all other agents alone (2.05 versus 1.06-1.36 log cfu decrease). Our findings raise concerns about the efficacy of commonly prescribed antibiotics against intracellular SA reservoirs and emphasize the need to consider targeting pathogen eradication from the liver to achieve early control of SAB.

Genotypic Shift and Diversification of MRSA Blood Stream Isolates in a University Hospital Setting: Evidence from a 12-Year Observational Study.

There have been few reports regarding the long-term trends in the genotypes of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates. Therefore, this study was performed to investigate the longitudinal trends in the genotypes of MRSA bloodstream isolates obtained from hospitalized patients during a 12-year study period from 2010 to 2021 at a tertiary care university hospital. Over the 12-year period from 2010 to 2021, we conducted a genetic investigation focusing on 245 MRSA strains isolated from the blood of hospitalized patients. The genotypes of the MRSA bloodstream isolates were determined by Staphylococcal Cassette Chromosome mec (SCCmec) typing, accessory gene regulator (agr) typing, PCR-based ORF typing (POT), and multilocus sequence typing (MLST). Strains with the same POT type detected in two or more isolates were designated as epidemic clones, while strains without a common POT type were classified as sporadic clones. Until 2015, isolates with SCCmec II/agr II were prevalent, but isolates with SCCmec IV/agr III increased from 2016. A total of 128 strains (52%) were identified as epidemic clones, while 117 strains (48%) were classified as sporadic clones. The detection rate of sporadic clones increased significantly since 2016 (p < 0.05). The epidemic clones were classified into three clusters, with MRSA of clonal complex (CC) 1 being prominent after 2016. This study showed that the genotypes of MRSA bloodstream isolates underwent a shift from SCCmec II/agr II type to SCCmec IV/agr III type, with a notable increase in MRSA of CC1, after 2016. There was a significant increase in the proportion of sporadic strains among the isolates, suggesting the diversification of genotypes.

Demographic, clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli bloodstream infections in Central Australia

We describe the demographics, clinical and molecular epidemiology of extended-spectrum β-lactamase (ESBL) Escherichia coli bloodstream infections (BSI) in Central Australia. All ESBL-producing E.coli bloodstream isolates from January 2018 to December 2020 were retrospectively identified. Demographic and clinical information was extracted by chart review. Whole-genome sequencing was performed for multi-locus sequence typing, antibiotic-resistance genes, and phylogenetic relationships. We identified 41 non-duplicate episodes of ESBL E.coli BSI. Median age was 55 years (IQR 47-63), 78% were female, 93% were Aboriginal, and half came from a remote community. Infections were predominantly urinary (68%, 28/41). In the 12 months prior, 70% (26/37) of identified patients had been hospitalised and 81% (30/37) prescribed antibiotics. Meropenem and piperacillin-tazobactam susceptibility was maintained in 100% and 95% of isolates, respectively. Co-resistance to non-β-lactam antibiotics was 32% to gentamicin, 61% to trimethoprim/sulfamethoxazole, and 68% to ciprofloxacin. For sequenced isolates, 41% (16/35) were sequence type 131 (ST131). Mean acquired antibiotic-resistance genes for each isolate was 12.3 (SD 3.1). Four isolates carried an OXA-1 gene. Only non-ST131 isolates carried AmpC and acquired quinolone-resistance genes. There was some evidence of clustering of closely related strains, but no evidence of community or healthcare admission overlap. ESBL rates are rapidly rising in Central Australia, which is a conducive environment for antibiotic resistance development (e.g., overcrowding, socioeconomic disadvantages, high healthcare exposure and high antibiotic use). Future research is required to explore resistance-transmission dynamics in this unique setting.

Candida auris – Comparison of sensititre YeastOne and Vitek 2 AST systems for antifungal susceptibility testing – A single centre experience

IntroductionCandida auris is emerging as an important cause of candidemia and deep seated candidal infection. We compared the susceptibility results of bloodstream Candida auris isolates by Vitek 2 with Sensititre YeastOne (SYO) method. MethodsForty-seven C. auris blood stream isolates were simultaneously tested for AFST by Vitek 2 and SYO. ResultsAll strains were resistant to Fluconazole. 25.5% isolates showed pan-azole resistance. In comparison with SYO, lower MICs for voriconazole were noted with Vitek 2 (VME rate 76.1%). All strains were sensitive to anidulafungin and micafungin by SYO. For micafungin, Vitek 2 demonstrated higher MICs and an ME rate of 23.5%. Susceptibility interpretation of caspofungin by SYO was challenged by development of ‘Eagle effect’ resulting in sensitivity of 28.2%. We studied the evolution of caspofungin ‘Eagle effect’ with SYO by serial hourly MIC readings and noted that paradoxical growth commenced at 21 hrs of incubation. Compared to SYO, Vitek 2 showed higher resistance rate to Amphotericin B with ME rate of 25.6%. ConclusionLaboratories using commercial AFST systems for Candida auris need to be aware of the possibility of ME and VME for amphotericin B and voriconazole respectively with Vitek 2 and ‘Eagle effect’ for caspofungin with SYO.

Different sampling strategies for optimal detection of the overall genetic diversity of methicillin-resistant Staphylococcus aureus.

Surveillance schemes for methicillin-resistant Staphylococcus aureus (MRSA) are widely established at the national and international levels. Due to the simple standardization of the protocol, mainly isolates from bloodstream infections are used. However, the limitations of this simple surveillance system are well described. We conducted a comprehensive analysis of MRSA isolates in a large Slovenian region over 5 years to identify the optimal sample group for assessing the overall MRSA diversity. At the same time, this study provides to date non-available molecular characterization of Slovenian MRSA isolates. A total of 306 MRSA isolates from various sources were sequenced and phenotypically tested for resistance. The isolates exhibited significant molecular diversity, encompassing 30 multi locus sequence type (MLST) sequence types (STs), 39 ST-SCCmec genetic lineages, 49 spa types, and 29 antibiotic resistance profiles. Furthermore, the isolate pool comprised 57 resistance genes, representing 22 resistance mechanisms, and 96 virulence genes. While bloodstream isolates, commonly used in surveillance, provided insights into frequently detected clones, they overlooked majority of clones and important virulence and resistance genes. Blood culture isolates detected 21.3% spa types, 24.1% resistance phenotypes, and 28.2% MLST-SCCmec profiles. In contrast, strains from soft tissues demonstrated superior genomic diversity capture, with 65.3% spa types, 58.6% resistance phenotypes, and 71.8% MLST-SCCmec profiles. These strains also encompassed 100.0% of virulence and 82.5% of resistance genes, making them better candidates for inclusion in surveillance programs. This study highlights the limitations of relying solely on bloodstream isolates in MRSA surveillance and suggests incorporating strains from soft tissues to obtain a more comprehensive understanding of the epidemiology of MRSA.IMPORTANCEIn this study, we investigated the diversity of methicillin-resistant Staphylococcus aureus (MRSA), a bacterium that can cause infections that are difficult to treat due to its resistance to antimicrobial agents. Currently, surveillance programs for MRSA mainly rely on isolates from bloodstream infections, employing a standardized protocol. However, this study highlights the limitations of this approach and introduces a more comprehensive method. The main goal was to determine which group of samples is best suited to understand the overall diversity of MRSA and to provide, for the first time, molecular characterization of Slovenian MRSA isolates. Our results suggest that including MRSA strains from soft tissue infections rather than just blood infections provides a more accurate and comprehensive view of bacterial diversity and characteristics. This insight is valuable for improving the effectiveness of surveillance programs and for developing strategies to better manage MRSA infections.

Dissemination of meticillin-resistant Staphylococcus aureus sequence type 8 (USA300) in Taiwan

BackgroundIn Taiwan, sequence type (ST) 239 and ST59 were two major clones among methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates in the past two decades. USA300 (ST8) prevailed in Americas but not in outside areas. Recently USA300 (ST8) emerged and were increasingly identified in Taiwan; we thus conducted an islandwide study to explore the role of USA300 among MRSA isolates. Methods100 MRSA bloodstream isolates identified in 2020, respectively, from each of the six participating hospitals in Taiwan were collected and characterized. First ten ST8 isolates from each hospital were further analyzed by whole genome sequencing (WGS). ResultsOf the 590 confirmed MRSA isolates, a total of 22 pulsotypes and 21 STs were identified. The strain of pulsotype AI/ ST8 was the most common lineage identified, accounting for 187 isolates (31.7%) and dominating in 5 of 6 hospitals, followed by pulsotype A/ ST239 (14.7%), pulsotype C/ ST59 (13.9%) and pulsotype D/ ST59 (9.2%). Of the 187 pulsotype AI/ ST8 isolates, 184 isolates were characterized as USA300 and clustered in three major sub-pulsotypes, accounting for 78%. Ninety percent of the 60 ST8 isolates for WGS were clustered in three major clades. ConclusionsIn 2020, USA300 became the most common clone of MRSA in Taiwan, accounting for > 30% of MRSA bloodstream isolates islanwide. Most of USA300 isolates circulating in Taiwan might be imported at multiple occasions and evolved into at least 3 successful local clades. MRSA USA300 had successfully established its role in Taiwan, an area outside of Americas.

Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

Genotypic and phenotypic characteristics of Candida parapsilosis bloodstream isolates: Health Care Associated Infections in a teaching Hospital in Italy

BackgroundCandidemia is the most common healthcare associated invasive fungal infection. Over the last few decades, candidemia caused by Candida species other than Candida albicans, particularly the Candida parapsilosis complex, has emerged worldwide. The aims of this study were: to analyze the genotypic and phenotypic characteristics of C. parapsilosis strains isolated from blood cultures and the environment in a hospital in southern Italy, to study the possible source of infection and to correlate the isolated strains. MethodsFrom April to October 2022, cases of candidemia due to C. parapsilosis in patients admitted to a hospital in the Apulia region were investigated. However, 119 environmental samples from the intensive care unit were collected for identification of the likely environmental reservoir of infection. Routine antifungal (amphotericin B, anidulafungin, fluconazole) susceptibility was performed on all isolates. Whole genome sequencing was performed to study the genotypic correlation of the isolates. Biofilm biomass and metabolic activity were also quantified for all isolates. ResultsA total of 43 C. parapsilosis isolates were cultured from the bloodstream of each patient in different departments, and seven surface samples were positive for C. parapsilosis. Most of the isolated yeasts (41/50; 85 %) were resistant to fluconazole and were genetically related to each other, suggesting an ongoing clonal outbreak of this pathogen. The fluconazole-susceptible isolates produced significantly more biofilm than did the resistant isolates. Metabolic activity was also higher for fluconazole-susceptible than resistant isolates. ConclusionCross-transmission of the microorganisms is suggested by the phenotypic similarity and genetic correlation between clinical and environmental strains observed in our study.

Bacterial etiology and antimicrobial resistance pattern of pediatric bloodstream infections: a 5-year experience in an Iranian referral hospital

BackgroundBloodstream infections (BSI) are the major cause of morbidity and mortality in children in developing countries. The purpose of the current study was to establish the antimicrobial susceptibility pattern of bacterial isolates from bloodstream infections at Children’s Medical Center Hospital (CMC), Tehran, Iran.MethodsWe retrospectively recorded all positive blood cultures and antimicrobial susceptibility of all bloodstream isolates among children admitted to CMC, during 5 years. Specimen culture, bacterial identification, and antimicrobial susceptibility testing were performed according to standard laboratory methods.ResultsFrom 3,179 pathogens isolated from the blood cultures 2,824 bacteria were cultured, with 1,312 cases being identified as Gram-positive bacteria (46%) and 1,512 cases as Gram-negative bacteria (54%). The most common Gram-negative bacteria isolated were as follows: Pseudomonas spp. (n = 266, 17.6%), Klebsiella pneumoniae (n = 242, 16%), Stenotrophomonas maltophilia (n = 204, 13.5%), Enterobacter spp. (n = 164, 10.8%), Escherichia coli (n = 159, 10.5%), Pseudomonas aeruginosa (n = 126, 8.3%), Serratia marcescens (n = 121, 8%), and Acinetobacter baumannii (n = 73, 4.8%). The most common Gram-positive bacteria isolated were coagulase-negative staphylococci (CONS) (n = 697, 53%), Streptococcus spp. (n = 237, 18%), Staphylococcus aureus (n = 202, 15%) and Enterococcus spp. (n = 167, 12.7%). 34% of bacterial strains were isolated from ICUs. The rates of methicillin resistance in S. aureus and CONS were 34% and 91%, respectively. E. coli isolates showed high resistance to cefotaxime (84%). All isolates of K. pneumoniae were susceptible to colistin and 56% were susceptible to imipenem. P. aeruginosa isolates showed high susceptibility to all antibiotics.ConclusionsOur findings emphasize the need of clinicians having access to up-to-date bacterial susceptibility data for routinely prescribed drugs. Continuous monitoring of changes in bacterial resistance will aid in the establishment of national priorities for local intervention initiatives in Iran. The increased risk of BSI caused by antibiotic-resistant organisms, emphasizes the significance of implementing appropriate antibiotic prescribing regulations and developing innovative vaccination techniques in Iran.

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Heteroresistance to piperacillin/tazobactam in Klebsiella pneumoniae is mediated by increased copy number of multiple β-lactamase genes.

Piperacillin/tazobactam is a β-lactam/β-lactamase inhibitor combination with a broad spectrum of activity that is often used as empirical and/or targeted therapy among hospitalized patients. Heteroresistance (HR) is a form of antibiotic resistance in which a minority population of resistant cells coexists with a majority susceptible population that has been found to be a cause of antibiotic treatment failure in murine models. To determine the prevalence of HR and mechanisms of HR to piperacillin/tazobactam among Klebsiella pneumoniae bloodstream infection (BSI) isolates. From July 2018 to June 2021, K. pneumoniae piperacillin/tazobactam-susceptible BSI isolates were collected from two tertiary hospitals in Atlanta, GA, USA. Only first isolates from each patient per calendar year were included. Population analysis profiling (PAP) and WGS were performed to identify HR and its mechanisms. Among 423 K. pneumoniae BSI isolates collected during the study period, 6% (25/423) were found to be HR with a subpopulation surviving above the breakpoint. WGS of HR isolates grown in the presence of piperacillin/tazobactam at concentrations 8-fold that of the MIC revealed copy number changes of plasmid-located β-lactamase genes blaCTX-M-15, blaSHV33, blaOXA-1 and blaTEM-1 by tandem gene amplification or plasmid copy number increase. Prevalence of HR to piperacillin/tazobactam among bloodstream isolates was substantial. The HR phenotype appears to be caused by tandem amplification of β-lactamase genes found on plasmids or plasmid copy number increase. This raises the possibility of dissemination of HR through horizontal gene transfer and requires further study.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.