Pseudomonas aeruginosa is a rod-shaped Gram-negative bacterium which is notably known as a pathogen in humans, animals, and plants. Infections caused by P. aeruginosa especially in hospitalized patients are often life-threatening and rapidly increasing worldwide throughout the years. Recently, multidrug-resistant P. aeruginosa has taken a toll on humans' health due to the inefficiency of antimicrobial agents. Therefore, the rapid and advanced diagnostic techniques to accurately detect this bacterium particularly in clinical samples are indeed necessary to ensure timely and effective treatments and to prevent outbreaks. This review aims to discuss most recent of state-of-the-art molecular diagnostic techniques enabling fast and accurate detection and identification of P. aeruginosa based on well-developed genotyping techniques, e.g., polymerase chain reaction, pulse-field gel electrophoresis, and next generation sequencing. The advantages and limitations of each of the methods are also reviewed.

Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI), GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101-104 copies/mL-sewage, 105-108 copies/g-human feces, and 102-105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

With the decline in the cost of whole-genome sequencing because of the introduction of next-generation sequencing (NGS) techniques, many public health and clinical laboratories have started to use bacterial whole genomes for epidemiological surveillance and clinical investigation. For epidemiological and clinical purposes in this "NGS era," whole-genome-scale single nucleotide polymorphism (wgSNP) analysis for genotyping is considered suitable. In this paper, we present an online service, PathoBacTyper (http://halst.nhri.org.tw/PathoBacTyper/), for pathogenic bacteria identification and genotyping based on wgSNP analysis. More than 400 pathogenic bacteria can be identified and genotyped through this service. Four data sets containing 59 Salmonella Heidelberg isolates from three outbreaks with the same pulsed-field gel electrophoresis pattern, 34 Salmonella Typhimurium isolates from six outbreaks, 103 isolates of hospital-associated vancomycin-resistant Enterococcus faecium and 15 Legionella pneumophila isolates from clinical and environmental samples in Israel were used for demonstrating the operation and testing the performance of the PathoBacTyper service. The test results reveal the applicability of this service for epidemiological typing and clinical investigation.

Toxoplasmosis is a zoonotic food-borne disease caused by Toxoplasma gondii, a land-derived protozoan parasite that infects a broad range of terrestrial and aquatic hosts. T. gondii may reach coastal waters via contaminated freshwater runoff and its oocysts may enter into the marine food web. Marine invertebrates as mussels being filter feeders are exposed and may concentrate T. gondii oocysts representing a potential source of infection for animals and humans. The present works investigated the prevalence, parasite burden and genotypes of T. gondii in the Mediterranean mussels (Mytilus galloprovincialis) from southern Italy. We sampled a total of 382 individual Mediterranean mussels from May to August 2018 from seven production sites in the Gulf of Naples (Campania region). An additional sample including 27 farmed Mediterranean mussels was obtained in February 2018 from a mollusk depuration plant in Corigliano Calabro (Calabria region). T. gondii DNA was detected in 43 out of 409 (10.5%) Mediterranean mussels from seven out of eight sampling sites. The number of T. gondii copies/g in the digestive gland ranged from 0.14 to 1.18. Fragment analysis of Short Tandem Repeats (STRs) at 5 microsatellite loci was performed from 10 T. gondii PCR positive samples revealing the presence of five distinct genotypes including one corresponding to type I and four atypical genotypes. These findings suggest potential implications of epidemiological importance for human and animal health because both type I and atypical genotypes could be highly pathogenic.

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.

The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K-antigen and colanic acid (CA) biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

Bacteria rapidly change their transcriptional patterns during infection in order to adapt to the host environment. To investigate host-bacteria interactions, various strategies including the use of animal infection models, in vitro assay systems and microscopic observations have been used. However, these studies primarily focused on a few specific genes and molecules in bacteria. High-density tiling arrays and massively parallel sequencing analyses are rapidly improving our understanding of the complex host-bacterial interactions through identification and characterization of bacterial transcriptomes. Information resulting from these high-throughput techniques will continue to provide novel information on the complexity, plasticity, and regulation of bacterial transcriptomes as well as their adaptive responses relative to pathogenecity. Here we summarize recent studies using these new technologies and discuss the utility of transcriptome analysis.

Although climate change is predicted to affect methane (CH4) emissions in paddy soil, the dynamics of methanogens and methanotrophs in paddy fields under climate change have not yet been fully investigated. To address this issue, a multifactor climate change experiment was conducted in a Chinese paddy field using the following experimental treatments: (1) enrichment of atmospheric CO2 concentrations (500 ppm, CE), (2) canopy air warming (2°C above the ambient, WA), (3) combined CO2 enrichment and warming (CW), and (4) ambient conditions (CK). We analyzed the abundance of methanogens and methanotrophs, community structures, CH4 production and oxidation potentials, in situ CH4 emissions using real-time PCR, T-RFLP, and clone library techniques, as well as biochemical assays. Compared to the control under CE and CW treatments, CH4 production potential, methanogenic gene abundance and soil microbial biomass carbon significantly increased; the methanogenic community, however, remained stable. The canopy air warming treatment only had an effect on CH4 oxidation potential at the ripening stage. Phylogenic analysis indicated that methanogens in the rhizosphere were dominated by Methanosarcina, Methanocellales, Methanobacteriales, and Methanomicrobiales, while methanotrophic sequences were classified as Methylococcus, Methylocaldum, Methylomonas, Methylosarcina (Type I) and Methylocystis (Type II). However, the relative abundance of Methylococcus (Type I) decreased under CE and CW treatments and the relative abundance of Methylocystis (Type II) increased. The in situ CH4 fluxes indicated similar seasonal patterns between treatments; both CE and CW increased CH4 emissions. In conclusion results suggest that methanogens and methanotrophs respond differently to elevated atmospheric CO2 concentrations and warming, thus adding insights into the effects of simulated global climate change on CH4 emissions in paddy fields.

The food industry is facing a major transition regarding methods for confirmation, characterization, and subtyping of Salmonella. Whole-genome sequencing (WGS) is rapidly becoming both the method of choice and the gold standard for Salmonella subtyping; however, routine use of WGS by the food industry is often not feasible due to cost constraints or the need for rapid results. To facilitate selection of subtyping methods by the food industry, we present: (i) a comparison between classical serotyping and selected widely used molecular-based subtyping methods including pulsed-field gel electrophoresis, multilocus sequence typing, and WGS (including WGS-based serovar prediction) and (ii) a scoring system to evaluate and compare Salmonella subtyping assays. This literature-based assessment supports the superior discriminatory power of WGS for source tracking and root cause elimination in food safety incident; however, circumstances in which use of other subtyping methods may be warranted were also identified. This review provides practical guidance for the food industry and presents a starting point for further comparative evaluation of Salmonella characterization and subtyping methods.

Staphylococcus aureus (S. aureus) constantly evolves under host and environment pressures. The monitoring network is essential in assessing the epidemiology of S. aureus infections. A total of 555 S. aureus isolates were collected from five hospitals in three different geographical regions of China for the investigation of molecular characteristics, antibiotic resistance, virulence gene, and wall teichoic acid (WTA) glycosyltransferase gene profiles. 233 (42.0%) isolates were identified as MRSA, and 323 (58.2%) were defined as multidrug-resistant (MDR) isolates. MRSA prevalence showed no significant difference among the three regions. In contrast, the MDR prevalence was significantly higher in central China than that in northern China (63.5% vs. 50.8%, P < 0.05). Thirty-eight sequence types (STs) belonging to 17 clone complexes (CCs) and 126 distinct spa-types were identified. The most prevalent clone was ST59-t437 (9.7%, 54/555), followed by ST22-t309 (7.6%, 42/555) and ST5-t2460 (7.2%, 40/555). Most ST59-t437 and ST5-t2460 were MRSA isolates, whereas most ST22-t309 was MSSA isolates. The predominant clones varied in different geographical areas. The distribution of the pvl, etb, tsst, clfb, sdrC, sdrD, hlg, fnbA, and hla genes showed significant differences among different regions. We found five WTA glycosyltransferase gene profiles, with tarP-/tarS+/tarM-/tagN- being the most common combination. Remarkably, the tarP gene was identified in more CCs than just CC5 and CC398. All of 16 tarP-positive isolates also contained the tarS. Moreover, tarS was present in almost all S. aureus isolates except 10 ST630 isolates. The tagN gene was only detected in 10 of 12 ST630 S. aureus isolates without tarS. The tarM gene was absent in CC5 and CC398. In brief, there were regional differences among molecular characteristics, antibiotic resistance, and virulence gene profiles. The tarS-negative ST630 lineage carried the tagN, which was never found before, indicating that it may be capable of expressing GroP-α-GalNAc WTA and exchanging mobile genetic elements with coagulase-negative staphylococci (CoNS).

Fourier transform infrared (FTIR) spectroscopy, a technology traditionally used in chemistry to determine the molecular composition of a wide range of sample types, has gained growing interest in microbial typing. It is based on the different vibrational modes of the covalent bonds between atoms of a given sample, as bacterial cells, induced by the absorption of infrared radiation. This technique has been largely used for the study of pathogenic species, especially in the clinical field, and has been proposed also for the typing at different subspecies levels. The high throughput, speed, low cost, and simplicity make FTIR spectroscopy an attractive technique also for industrial applications, in particular, for probiotics. The aim of this study was to compare FTIR spectroscopy with established genotyping methods, pulsed-field gel electrophoresis (PFGE), whole-genome sequencing (WGS), and multilocus sequence typing (MLST), in order to highlight the FTIR spectroscopy potential discriminatory power at strain level. Our study focused on bifidobacteria, an important group of intestinal commensals generally recognized as probiotics. For their properties in promoting and maintaining health, bifidobacteria are largely marketed by the pharmaceutical, food, and dairy industries. Strains belonging to Bifidobacterium longum subsp. longum and Bifidobacterium animalis subsp. lactis were taken into consideration together with some additional type strains. For B. longum subsp. longum, it was possible to discriminate the strains with all the methods used. Although two isolates were shown to be strictly phylogenetically related, constituting a unique cluster, based on PFGE, WGS, and MLST, no clustering was observed with FTIR. For B. animalis subsp. lactis group, PFGE, WGS, and MLST were non-discriminatory, and only one strain was easily distinguished. On the other hand, FTIR discriminated all the isolates one by one, and no clustering was observed. According to these results, FTIR analysis is not only equivalent to PFGE, WGS, and MLST, but also for some strains, in particular, for B. animalis subsp. lactis group, more informative, being able to differentiate strains not discernible with the other two methods based on phenotypic variations likely deriving from certain genetic changes. Fourier transform infrared spectroscopy has highlighted the possibility of using the cell surface as a kind of barcode making tracing strains possible, representing an important aspect in probiotic applications. Furthermore, this work constitutes the first investigation on bifidobacterial strain typing using FTIR spectroscopy.

Hypervirulent strains of Klebsiella pneumoniae (hvKP) are genetic variants of K. pneumoniae which can cause life-threatening community-acquired infection in healthy individuals. Currently, methods for efficient differentiation between classic K. pneumoniae (cKP) and hvKP strains are not available, often causing delay in diagnosis and treatment of hvKP infections. To address this issue, we devised a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) approach for rapid identification of K1 hvKP strains. Four standard algorithms, genetic algorithm (GA), support vector machine (SVM), supervised neural network (SNN), and quick classifier (QC), were tested for their power to differentiate between K1 and non-K1 strains, among which SVM was the most reliable algorithm. Analysis of the receiver operating characteristic curves of the interest peaks generated by the SVM model was found to confer highly accurate detection sensitivity and specificity, consistently producing distinguishable profiles for K1 hvKP and non-K1 strains. Of the 43 K. pneumoniae modeling strains tested by this approach, all were correctly identified as K1 hvKP and non-K1 capsule type. Of the 20 non-K1 and 17 K1 hvKP validation isolates, the accuracy of K1 hvKP and non-K1 identification was 94.1 and 90.0%, respectively, according to the SVM model. In summary, the MALDI-TOF MS approach can be applied alongside the conventional genotyping techniques to provide rapid and accurate diagnosis, and hence prompt treatment of infections caused by hvKP.

The yeast species Brettanomyces bruxellensis is associated with important economic losses due to red wine spoilage. The most common method to prevent and/or control B. bruxellensis spoilage in winemaking is the addition of sulfur dioxide into must and wine. However, recently, it was reported that some B. bruxellensis strains could be tolerant to commonly used doses of SO2. In this work, B. bruxellensis response to SO2 was assessed in order to explore the relationship between SO2 tolerance and genotype. We selected 145 isolates representative of the genetic diversity of the species, and from different fermentation niches (roughly 70% from grape wine fermentation environment, and 30% from beer, ethanol, tequila, kombucha, etc.). These isolates were grown in media harboring increasing sulfite concentrations, from 0 to 0.6 mg.L-1 of molecular SO2. Three behaviors were defined: sensitive strains showed longer lag phase and slower growth rate and/or lower maximum population size in presence of increasing concentrations of SO2. Tolerant strains displayed increased lag phase, but maximal growth rate and maximal population size remained unchanged. Finally, resistant strains showed no growth variation whatever the SO2 concentrations. 36% (52/145) of B. bruxellensis isolates were resistant or tolerant to sulfite, and up to 43% (46/107) when considering only wine isolates. Moreover, most of the resistant/tolerant strains belonged to two specific genetic groups, allowing the use of microsatellite genotyping to predict the risk of sulfur dioxide resistance/tolerance with high reliability (>90%). Such molecular diagnosis could help the winemakers to adjust antimicrobial techniques and efficient spoilage prevention with minimal intervention.

Streptococcus agalactiae (group B Streptococcus, GBS) is one of the most important agents of bovine mastitis and causes remarkable direct and indirect economic losses to the livestock sector. Moreover, this species can cause severe human diseases in susceptible individuals. To investigate the zoonotic potential of S. agalactiae, 203 sympatric isolates from both humans and cattle, isolated in the same time frame (2018) and in the same geographic area (Emilia Romagna region, Northern Italy), were characterized by molecular capsular typing (MCT), pilus island typing (PI), and multi-locus sequence typing (MLST). In addition, antibiotic-resistant phenotypes were investigated. The distribution of the allelic profiles obtained by combining the three genotyping methods (MCT-PI-MLST) resulted in 64 possible genotypes, with greater genetic variability among the human compared to the bovine isolates. Although the combined methods had a high discriminatory power (>96,2%), five genotypes were observed in both species (20,9% of the total isolates). Furthermore, some of these strains shared the same antibiotic resistance profiles. The finding of human and bovine isolates with common genotypes and antibiotic resistance profiles supports the hypothesis of interspecies transmission of S. agalactiae between bovines and humans.

Objectives: Sequence type 1193 is emerging as a new, virulent and resistant lineage among fluoroquinolone resistant Escherichia coli (FQrE. coli). In this study, we investigated the prevalence and molecular characteristics of this clone isolated from a Chinese university hospital. Methods: 73 phylogenetic group B2-FQr-non-ST131 isolates were collected from August 2014 and August 2015 at a Chinese university hospital. Isolates were screened for ST1193 by multilocus sequence typing. E. coli ST1193 then underwent lactose fermentation determination, susceptibility testing, virulence genotyping, PCR-based O typing, pulsed-field gel electrophoresis (PFGE) and FQr mechanism analysis. Results: Of 73 B2-FQr-non ST131 E. coli isolates, 69.9% (n = 51) were ST1193. 90.2% (46/51) of ST1193 isolates were O75 serotype and 96.1% (49/51) of the ST1193 isolates were lactose non-fermenters. 35 clusters were identified by PFGE. ST1193 isolates exhibited a set of 3 conserved mutations defining quinolone-resistance determining region substitutions (gyrA S83L, D87N, and parC S80I). The most frequent VF genes detected in these E. coli ST1193 isolates were fyuA (yersiniabactin, 96.1%), fimH (type 1 fimbriae, 94.1%), iutA (iron uptake gene, 90.2%), kpsMT II (group II capsule, 90.2%), kpsK1 (K1 capsule, 86.3%) and PAI. Conclusion: ST1193 lineage accounts for the majority of group B2-FQr-non-ST131 E. coli clinical isolates. Most of the ST1193 are serotype O75 and lactose non-fermenting. Strategic surveillance and control schemes are needed in the future for this newly emerging clone of E. coli: B2-FQr-ST1193.

Cronobacter sakazakii is an opportunistic pathogen that causes severe infections in neonates and infants through contaminated powdered infant formula (PIF). Therefore, the aim of this study was a large-scale study on determine the prevalence, molecular characterization and antibiotic susceptibility of C. sakazakii isolates from PIF purchased from Chinese retail markets. Two thousand and twenty PIF samples were collected from different institutions. Fifty-six C. sakazakii strains were isolated, and identified using fusA sequencing analysis, giving a contamination rate of 2.8%. Multilocus sequence typing (MLST) was more discriminatory than other genotyping methods. The C. sakazakii isolates were divided into 14 sequence types (STs) by MLST, compared with only seven clusters by ompA and rpoB sequence analysis, and four C. sakazakii serotypes by PCR-based O-antigen serotyping. C. sakazakii ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) were the dominant sequence types isolated. C. sakazakii serotype O2 (34/56, 60.7%) was the primary serotype, along with ompA6 and rpoB1 as the main allele profiles, respectively. Antibiotic susceptibility testing indicated that all C. sakazakii isolates were susceptible to ampicillin-sulbactam, cefotaxime, ciprofloxacin, meropenem, tetracycline, piperacillin-tazobactam, and trimethoprim-sulfamethoxazole. The majority of C. sakazakii strains were susceptible to chloramphenicol and gentamicin (87.5 and 92.9%, respectively). In contrast, 55.4% C. sakazakii strains were resistant to cephalothin. In conclusion, this large-scale study revealed the prevalence and characteristics of C. sakazakii from PIF in Chinese retail markets, demonstrating a potential risk for neonates and infants, and provide a guided to effective control the contamination of C. sakazakii in production process.

Thermophilic Campylobacter species are among the major etiologies of bacterial enteritis globally. This study aimed at assessing the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic Campylobacter species isolated from a layer poultry farm in South Korea. One hundred fifty-three chicken feces were collected from two layer poultry farms in Gangneung, South Korea. The Campylobacter species were isolated by cultural techniques, while PCR and sequencing were used for species confirmation. Antimicrobial susceptibility testing for six antimicrobials [ciprofloxacin (CIP), nalidixic acid (NAL), sitafloxacin (SIT), erythromycin (ERY), tetracycline (TET), and gentamicin (GEN)] was carried out by broth microdilution. Three AMR and nine virulence genes were screened by PCR. Genotyping was performed by flaA-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Of the 153 samples, Campylobacter spp. were detected in 55 (35.9%), with Campylobacter jejuni and Campylobacter coli being 49 (89.1%) and six (10.9%), respectively. High-level resistance was observed for CIP (100%), NAL (100%), and TET (C. jejuni, 93.9%; C. coli: 83.3%). No resistance was observed for SIT. The missense mutation (C257T) in gyrA gene was confirmed by sequencing, while the tet(O) gene was similar to known sequences in GenBank. The rate of multidrug-resistant (MDR) strains was 8.2%, and they all belonged to C. jejuni. All Campylobacter isolates possessed five virulence genes (cdtB, cstII, flaA, cadF, and dnaJ), but none possessed ggt, while the rates for other genes (csrA, ciaB, and pldA) ranged between 33.3 and 95.9%. The flaA-RFLP yielded 26 flaA types (C. jejuni: 21 and C. coli: five), while the MLST showed 10 sequence types (STs) for C. jejuni and three STs for C. coli, with CC-607 (STs 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of C. jejuni, three were newly assigned. The findings of this study highlight the increased resistance to quinolones and TET, the virulence potential, and the diverse genotypes among Campylobacter strains isolated from the layer poultry farm.

Introduction: The increasing incidence of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in sub-Saharan Africa is of serious concern. Studies from countries with a highly industrialized poultry industry suggest the poultry production-food-consumer chain as a potential transmission route. In Africa, integrated studies at this human-animal interface are still missing. Aim: To determine the molecular epidemiology of ESBL-producing E. coli from the intestinal tract of humans and poultry in rural Ghana. Methods: During a 6-month period, fecal samples from all children admitted to the Agogo Hospital (Ghana) and broilers at eight poultry farms located within the hospital catchment area were collected. After screening on selective ESBL agar, whole genome sequencing (WGS) was performed on all ESBL isolates. The genomes were analyzed using multilocus sequence typing (MLST), ESBL genotyping and genome-based phylogenetic analyses. Results: Of 140 broilers and 54 children, 41 (29%) and 33 (61%) harbored ESBL E. coli, respectively, with prevalences on farms ranging between 0 and 85%. No predominant sequence type (ST) was detected among humans. ST10 was most prevalent among broilers (n = 31, 69%). The ESBL gene bla CTX-M-15 was predominant among broilers (n = 43, 96%) and humans (n = 32, 97%). Whole-genome-based phylogenetic analysis revealed three very closely related broiler/human isolate clusters (10% of ESBL isolates) with chromosomal and plasmid-mediated ESBL genes. Conclusion: The findings demonstrate a high frequency of intestinal ESBL-producing E. coli in rural Ghana. Considering that animal and human samples are independent specimens from the same geographic location, the number of closely related ESBL isolates circulating across these two reservoirs is substantial. Hence, poultry farms or meat products might be an important source for ESBL-producing bacteria in rural Ghana leading to difficult-to-treat infections in humans.

Vulvovaginal candidiasis (VVC) is a yeast infection with a global reach and millions of dollars are spent annually for its diagnosis and treatment. Recently, Candida glabrata with different degrees of antifungal resistance has been considered as the second most common cause of vaginal infections. The aim of the present study is to determine the antifungal susceptibility and molecular epidemiology profiles of C. glabrata isolates from patients with VVC. Sixty-one C. glabrata isolates were examined for antifungal susceptibility using the EUCAST broth microdilution method. Moreover, microsatellite length polymorphism (MLP) was used for typing the C. glabrata isolates using six microsatellite markers. Overall, 13, 3.3, and 0% of the isolates were non-wild types to itraconazole, posaconazole, and voriconazole, respectively. Sixty (98.4%) isolates were an intermediate phenotype to fluconazole and only one isolate was fluconazole resistant. Microsatellite length polymorphism with a discriminatory power of 0.964 identified 35 distinct types and 24 singleton genotypes. The assessment of the population genetic structure revealed that the non-wild-type population had a moderate genetic differentiation compared to the wild type population (FST = 0.1457). It was also found that the most common genotypes were G27 (eight strains), G12 (six strains), and G4 (five strains). We found that eight strains were resistant/a non-wild phenotype to itraconazole. Five out of eight (62.5%) resistant/non-wild phenotype strains correlated to a predominant genotype (GT27) and the rest belonged to GT11 (12.5%), GT29 (12.5%), and GT28 (12.5%). The current study is the first molecular epidemiology study in the southwest of Iran and demonstrates the antifungal susceptibility profiles of C. glabrata in it. This study shows a wide range of the genetic diversity of C. glabrata (35 different genotypes) from VVC in the southwest of Iran. The majority of the non-wild isolates had a dominant genotype or genotypes related to this dominant genotype (clonal cluster one).

The incidence of candidemia by the Candida parapsilosis complex has increased considerably in recent decades, frequently related to use of indwelling intravascular catheters. The ability of this pathogen to colonize healthcare workers (HCW)' hands, and to form biofilm on medical devices has been associated with the occurrence of nosocomial outbreaks and high mortality rates. Fluconazole has been the leading antifungal drug for the treatment of invasive candidiasis in developing countries. However, azole-resistant C. parapsilosis isolates are emerging worldwide, including in Brazil. Few studies have correlated outbreak infections due to C. parapsilosis with virulence factors, such as biofilm production. We thus conducted a microbiological investigation of C. parapsilosis complex isolates from a Brazilian teaching hospital. Additionally, we identified a previously unrecognized outbreak caused by a persistent azole-resistant C. parapsilosis (sensu stricto) clone in the intensive care unit (ICU), correlating it with the main clinical data from the patients with invasive candidiasis. The molecular identification of the isolates was carried out by PCR-RFLP assay; antifungal susceptibility and biofilm formation were also evaluated. The genotyping of all C. parapsilosis (sensu stricto) was performed by microsatellite analysis and the presence of ERG11 mutations was assessed in the azole non-susceptible isolates. Fourteen C. parapsilosis (sensu stricto) isolates were recovered from patients with invasive candidiasis, eight being fluconazole and voriconazole-resistant, and two intermediate only to fluconazole (FLC). All non-susceptible isolates showed a similar pattern of biofilm formation with low biomass and metabolic activity. The A395T mutation in ERG11 was detected exclusively among the azole-resistant isolates. According to the microsatellite analysis, all azole non-susceptible isolates from the adult ICU were clustered together indicating the occurrence of an outbreak. Regarding clinical data, all patients infected by the clonal non-susceptible isolates and none of the patients infected by the susceptible isolates had been previously exposed to corticosteroids (p = 0.001), while the remaining characteristics showed no statistical significance. The current study revealed the persistence of an azole non-susceptible C. parapsilosis clone with low capacity to form biofilm over two years in the adult ICU. These results reinforce the need of epidemiological surveillance and monitoring antifungal susceptibility of C. parapsilosis isolates in hospital wards.