Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

Introduction: Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups. Methods: Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients. Results: Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite. Conclusion: This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Analysis of virulence profiles in clinical isolates of Klebsiella pneumoniae from renal abscesses: clinical significance of hypervirulent isolates.

Introduction: Klebsiella pneumoniae can cause a wide range of infections. Hypervirulent K. pneumoniae (hvKp), particularly associated with the K1 and K2 capsular types, is an increasingly significant microorganism with the potential to cause invasive infections, including renal abscesses. Despite the rising prevalence of hvKp infections, information on renal abscesses caused by K. pneumoniae is limited, and the clinical significance of hvKp associated with specific virulence genes remains elusive. Methods: This study performed at a 1200-bed tertiary hospital sought to identify the clinical and microbiological characteristics of renal abscesses caused by K. pneumoniae, focusing on various virulence genes, including capsular serotypes and multilocus sequence typing (MLST). Results: Over an 8-year period, 64 patients with suspected renal abscesses were reviewed. Ten patients diagnosed with K. pneumoniae-related renal abscesses were ultimately enrolled in the study. Among the isolates from the 10 patients, capsular serotype K2 was predominant (40.0%), followed by K1 (30.0%). The most common sequence type by MLST was 23 (40.0%). In particular, six patients (60.0%) harbored specific genes indicative of hvKp: iucA, peg-344, rmpA, and rmpA2. Conclusions: Our findings highlight the importance of hvKp as a pathogen in renal abscesses. Although the nature of hvKp is relatively unknown, it is widely recognized as a highly virulent pathogen that can infect relatively healthy individuals of various ages and simultaneously cause infections at multiple anatomical sites. Therefore, when treating patients with K. pneumoniae-related renal abscesses, caution is necessary when considering the characteristics of hvKp, such as potential bacteremia, multi-organ abscess formation, and metastatic spread.

Emergence of eravacycline heteroresistance in carbapenem-resistant Acinetobacter baumannii isolates in China.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to almost all antibiotics. Eravacycline, a newer treatment option, has the potential to treat CRAB infections, however, the mechanism by which CRAB isolates develop resistance to eravacycline has yet to be clarified. This study sought to investigate the features and mechanisms of eravacycline heteroresistance among CRAB clinical isolates. A total of 287 isolates were collected in China from 2020 to 2022. The minimum inhibitory concentration (MIC) of eravacycline and other clinically available agents against A. baumannii were determined using broth microdilution. The frequency of eravacycline heteroresistance was determined by population analysis profiling (PAP). Mutations and expression levels of resistance genes in heteroresistant isolates were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR), respectively. Antisense RNA silencing was used to validate the function of eravacycline heteroresistant candidate genes. Twenty-five eravacycline heteroresistant isolates (17.36%) were detected among 144 CRAB isolates with eravacycline MIC values ≤4 mg/L while no eravacycline heteroresistant strains were detected in carbapenem-susceptible A. baumannii (CSAB) isolates. All eravacycline heteroresistant strains contained OXA-23 carbapenemase and the predominant multilocus sequence typing (MLST) was ST208 (72%). Cross-resistance was observed between eravacycline, tigecycline, and levofloxacin in the resistant subpopulations. The addition of efflux pump inhibitors significantly reduced the eravacycline MIC in resistant subpopulations and weakened the formation of eravacycline heteroresistance in CRAB isolates. The expression levels of adeABC and adeRS were significantly higher in resistant subpopulations than in eravacycline heteroresistant parental strains (P < 0.05). An ISAba1 insertion in the adeS gene was identified in 40% (10/25) of the resistant subpopulations. Decreasing the expression of adeABC or adeRS by antisense RNA silencing significantly inhibited eravacycline heteroresistance. In conclusion, this study identified the emergence of eravacycline heteroresistance in CRAB isolates in China, which is associated with high expression of AdeABC and AdeRS.

A prospective bacterial whole-genome-sequencing-based surveillance programme for comprehensive early detection of healthcare-associated infection transmission in paediatric oncology patients.

BACKGROUND: Bacterial whole-genome sequencing (WGS) and determination of genetic relatedness is an important tool for investigation of epidemiologically suspected outbreaks. AIM: This prospective cohort study evaluated a comprehensive, prospective bacterial WGS-based surveillance programme for early detection of transmission of most bacterial pathogens among patients at a paediatric oncology hospital. METHODS: Cultured bacterial isolates from clinical diagnostic specimens collected prospectively from both inpatient and outpatient encounters between January 2019 and December 2021 underwent routine WGS and core genome multi-locus sequence typing to determine isolates' relatedness. Previously collected isolates from January to December 2018 were retrospectively analysed for identification of prior or ongoing transmission. Multi-patient clusters were investigated to identify potential transmission events based on temporal and spatial epidemiological links and interventions were introduced. FINDINGS: A total of 1497 bacterial isolates from 1025 patients underwent WGS. A total of 259 genetically related clusters were detected, of which 18 (6.9%) multi-patient clusters involving 38 (3.7%) patients were identified. Sixteen clusters involved two patients each, and two clusters involved three patients. Following investigation, epidemiologically plausible transmission links were identified in five (27.8%) multi-patient clusters. None of the multi-patient clusters were suspected by conventional epidemiological surveillance. CONCLUSION: Bacterial WGS-based surveillance for early detection of hospital transmission detected several limited multi-patient clusters that were unrecognized by conventional epidemiological methods. Genomic surveillance helped efficiently focus interventions while reducing unnecessary investigations.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil.

OBJECTIVES: We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. METHODS: Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. RESULTS: High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. CONCLUSION: Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

A cluster of three extrapulmonary Mycobacterium abscessus infections linked to well-maintained water-based heater-cooler devices.

BACKGROUND: Various water-based heater-cooler devices (HCDs) have been implicated in nontuberculous mycobacteria outbreaks. Ongoing rigorous surveillance for healthcare-associated M. abscessus (HA-Mab) put in place following a prior institutional outbreak of M. abscessus alerted investigators to a cluster of 3 extrapulmonary M. abscessus infections among patients who had undergone cardiothoracic surgery. METHODS: Investigators convened a multidisciplinary team and launched a comprehensive investigation to identify potential sources of M. abscessus in the healthcare setting. Adherence to tap water avoidance protocols during patient care and HCD cleaning, disinfection, and maintenance practices were reviewed. Relevant environmental samples were obtained. Patient and environmental M. abscessus isolates were compared using multilocus-sequence typing and pulsed-field gel electrophoresis. Smoke testing was performed to evaluate the potential for aerosol generation and dispersion during HCD use. The entire HCD fleet was replaced to mitigate continued transmission. RESULTS: Clinical presentations of case patients and epidemiologic data supported intraoperative acquisition. M. abscessus was isolated from HCDs used on patients and molecular comparison with patient isolates demonstrated clonality. Smoke testing simulated aerosolization of M. abscessus from HCDs during device operation. Because the HCD fleet was replaced, no additional extrapulmonary HA-Mab infections due to the unique clone identified in this cluster have been detected. CONCLUSIONS: Despite adhering to HCD cleaning and disinfection strategies beyond manufacturer instructions for use, HCDs became colonized with and ultimately transmitted M. abscessus to 3 patients. Design modifications to better contain aerosols or filter exhaust during device operation are needed to prevent NTM transmission events from water-based HCDs.

Network pharmacology and experimental verification to explore the anti-superficial thrombophlebitis mechanism of Mailuo shutong pill.

ETHNOPHARMACOLOGICAL RELEVANCE: Mailuo shutong pill (MLST) has been widely used in clinical treatment of superficial thrombotic phlebitis (STP). Nevertheless, the major active components of MLST and the mechanism of synergistic action have not been reported. AIM OF THE STUDY: The present study aimed to evaluate the improving effects and the underlying mechanism of MLST on mannitol-induced STP in rabbits. MATERIAL AND METHODS: In this study, Ultrahigh-performance liquid chromatography electrospray ionization quadrupole-exactive orbitrap mass spectrometry (UHPLC-ESI-Q-Exactive-Orbitrap-MS) was used to analyze and identify the chemical composition of MLST and the prototype components absorbed into the blood. Then, according to the prototype components in serum, the targets and mechanisms of MLST were explored by applying network pharmacology. The rabbit model of STP was established by injecting 20% mannitol into bilateral auricular vein. The pathological changes of rabbit ear tissues, inflammatory factors, coagulation function and hemorheology were detected. In addition, molecular docking verified the interaction between the main active ingredient and the key target. Finally, the PI3K/AKT pathway and its regulated downstream pathways were verified by Western blot. RESULTS: A total of 96 MLST components and 53 prototypical components absorbed into the blood were successfully identified. Based on network pharmacology, PI3K/AKT pathway and 10 chemical components closely related to this pathway were obtained. Hematoxylin-eosin (HE) staining results indicated that MLST effectively improved of the pathological damage of ear tissues. MLST decreased levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and C-reactive protein (CRP). The expression of platelets (PLT) and fibrinogen concentration (FIB) was decreased, while prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged. In addition, the plasma viscosity and whole blood viscosity in the MLST groups were significantly decreased. The more important discovery was that the expressions of P-PI3K, VEGF, P-AKT, P-IκB-α, P-NF-κB, NLRP3, ASC, Cleaved IL-1ß and Cleaved Caspase-1 were effectively reversed after treatment with MLST. CONCLUSIONS: This study comprehensively analyzed and characterized the chemical composition of MLST and the prototypical components absorbed into the blood. This study strongly confirmed the pharmacodynamic effect of MLST on STP. More importantly, this pharmacodynamic effect was achieved through inhibition of the PI3K/AKT pathway and its regulated NF-κB and NLRP3 pathways.

Phenotypic and genetic characterization of hypervirulent Klebsiella pneumoniae in patients with liver abscess and ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) and pyogenic liver abscess (PLA) due to Klebsiella pneumoniae infection can trigger life-threatening malignant consequences, however, there are few studies on the strain-associated clinical pathogenic mechanisms between VAP and PLA. A total of 266 patients consist of 129 VAP and 137 PLA were included for analysis in this study. We conducted a comprehensive survey for the two groups of K. pneumoniae isolates, including phenotypic experiments, clinical epidemiology, genomic analysis, and instrumental analysis, i.e., to obtain the genomic differential profile of K. pneumoniae strains responsible for two distinct infection outcomes. We found that PLA group had a propensity for specific underlying diseases, especially diabetes and cholelithiasis. The resistance level of VAP was significantly higher than that of PLA (78.57% vs. 36%, P < 0.001), while the virulence results were opposite. There were also some differences in key signaling pathways of biochemical processes between the two groups. The combination of iucA, rmpA, hypermucoviscous phenotype, and ST23 presented in K. pneumoniae infection is more important and highly prudent for timely treatment. The present study may contribute a benchmark for the K. pneumoniae clinical screening, epidemiological surveillance, and effective therapeutic strategies.

Clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus in bone and joint infection among children.

OBJECTIVE: To investigate the characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) in bone and joint infection (BJI) among children. METHODS: A total of 338 patients diagnosed with BJI from 2013 to 2022 in Children's Hospital of Fudan University were enrolled. Demographic information, microbiology culture results and laboratory findings, including white blood counts (WBC), C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), and erythrocyte sedimentation rate (ESR) were collected and analyzed. MRSA was confirmed by antimicrobial susceptibility testing. Other MRSA-caused infections were randomly selected for comparison. Twenty-three virulence and antimicrobial resistance (AMR) genes were screened for MRSA strains. Multilocus sequence typing (MLST) and Staphylococcal protein A (spa) typing were performed using PCR amplification and sequencing. RESULTS: Of the identified pathogens in BJI, MRSA accounted for 21.0% (47/224). Patients with BJI had high levels of initial CRP, white blood cell count (WBC) and IL-6. ST59 (43.9%) and t437 (37.6%) were the main MRSA subtypes isolated from the children. The major genotypes in BJI were ST59-t437 (29.8%) and ST22-t309 (14.9%), with high carriage of hemolysins including hla (94.4-100%), hlb (66.2-93.3%), and hld (100%). Notably, Panton-Valentine leukocidin (pvl) had a high prevalence (53.3%) in ST22-t309-MRSA. Other virulence genes including tst, seg and sei were more commonly detected in ST22-t309-MRSA (40.0-46.7%) than in ST59-t437-MRSA (4.2-9.9%). High-carriage AMR genes in MRSA included aph(3')/III (66.7-80%), ermB (57.5-73.3%) and ermC (66.7-78.9%). MRSA presented high-resistance to erythromycin (52.0-100%) and clindamycin (48.0-92.5%), different genotypes displayed variation in their susceptibilities to antibiotics. CONCLUSIONS: The major MRSA genotype in BJI was ST59-t437, followed by ST22-t309, with a higher prevalence of the pvl gene. Continuous surveillance of pvl-positive ST22-t309-MRSA in pediatric BJI infections is thus required.

[Therapeutic effect and mechanism of Mailuo Shutong Pills on posterior limb swelling caused by femur fracture in rats based on intestinal flora and intestinal metabolism].

This study aimed to investigate the protective effect and underlying mechanism of Mailuo Shutong Pills(MLST) on posterior limb swelling caused by femur fracture in rats. The rats were randomly divided into a sham operation group, a model group, a low-dose MLST group(1.8 g·kg~(-1)·d~(-1)), a high-dose MLST group(3.6 g·kg~(-1)·d~(-1)), and a positive drug group(60 mg·kg~(-1)·d~(-1) Maizhiling Tablets). The femur in the sham operation group was exposed and the wound was sutured, while the other four groups underwent mechanical damage to cause femur fracture. The rats were treated with corresponding drugs by gavage 7 days before modeling and 5 days after modeling, while those in the sham operation group and the model group were given an equivalent dose of distilled water by gavage. Hematoxylin-eosin(HE) staining was used to detect the pathological injury of the posterior limb muscle tissues in rats, and the degree of hind limb swelling was measured. The enzyme-linked immunosorbent assay(ELISA) kit was used to detect the expression levels of interleukin-6(IL-6), interleukin-1ß(IL-1ß), and tumor necrosis factor-α(TNF-α) in the serum of rats in each group. The activity of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and glutathione peroxidase(GSH-Px) in rat serum was also measured. Western blot was used to detect the protein expression levels of heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), and nuclear transcription factor E2-related factor 2(Nrf2) in rat posterior limb muscle tissues. The changes in the intestinal flora and intestinal metabolites in rats were detected by 16S rDNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), respectively, to explore the underlying mechanism of MLST in treating posterior limb swelling caused by femur fracture in rats. Compared with the model group, MLST significantly improved the degree of posterior limb swelling in rats, reduced the levels of serum inflammatory factors, and alleviated oxidative stress injury. The HE staining results showed that the inflammatory infiltration in the posterior limb muscle tissues of rats in the MLST groups was significantly improved. Western blot results showed that MLST significantly increased the protein expression of HO-1, NQO1, and Nrf2 in rat posterior limb muscle tissues compared with the model group. The 16S rDNA sequencing results showed that MLST improved the disorder of intestinal flora in rats after femur fracture. The UPLC-MS/MS results showed that MLST significantly affected the bile acid biosynthesis and metabolism pathway in the intestine after femur fracture, and the Spearman analysis confirmed that the metabolite deoxycholic acid involved in bile acid biosynthesis was positively correlated with the abundance of Turicibacter. The metabolite cholic acid was positively correlated with the abundance of Papilibacter, Staphylococcus, and Intestinimonas. The metabolite lithocholic acid was positively correlated with Papilibacter and Intestinimonas. The above results indicated that MLST could protect against the posterior limb swelling caused by femur fracture in rats. This protective effect may be achieved by improving the pathological injury of the posterior limb muscle, reducing the expression levels of inflammatory and oxidative stress-related factors in serum, reducing the oxidative injury of the posterior limb muscle, improving intestinal flora, and balancing the biosynthesis of bile acids in the intestine.

Prevalence of carbapenemase producing Enterobacterales colonization and risk factor of clinical infection.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are global concerns in infection control, and the number of CPE outbreaks in hospitals is increasing despite the strengthening of contact precautions. This study aimed to confirm the prevalence and transition rate of CPE infection from stool surveillance culture and to identify the acquisition pathway of CPE. METHODS: This is a longitudinal review of patients with stool surveillance cultures at a tertiary center in Seoul, South Korea, from July 2018 to June 2020. Pulsed-field gel electrophoresis, multi-locus sequence typing, and whole genome sequencing were performed for carbapenemase-producing Klebsiella pneumoniae and Escherichia coli strains. RESULTS: Among 1620 patients who had undergone stool CPE surveillance cultures, only 7.1% of active surveillance at the Emergency Room (ER) and 4.4% of universal surveillance in the Intensive Care Unit (ICU) were stool CPE positive. The transition rates from stool carriers to clinical CPE infections were 29.4% in the ER and 31.3% in the ICU. However, it was significantly high (55.0%) in the initial stool CPE-negative ICU patients. Among the initial stool CPE-positive patients, hypertension (61% vs. 92.3%, P = 0.004), malignancy (28.8% vs. 53.8%, P = 0.027), and mechanical ventilation (25.4% vs. 53.8%, P = 0.011) were significant risk factors for clinical CPE infection. Molecular typing revealed that sequence type (ST) 307 and ST 395 were dominant in K. pneumoniae, and ST 410 was dominant in E. coli isolates. CONCLUSIONS: Active surveillance showed a higher detection rate than universal stool CPE screening, and one-third of positive stool CPE specimens ultimately developed subsquent clinical CPE infection. According to the molecular typing of the identified CPE strains, in-hospital spread prevailed over external inflow, and the transition rate to clinical CPE was particularly high in the ICU. Therefore, in order to control CPE propagation, not only active surveillance to block inflow from outside, but also continuous ICU monitoring within the hospital is necessary.

Genomic and phenotypic characterization of Salmonella enterica serovar Kentucky.

Non-typhoidal Salmonella are extremely diverse and different serovars can exhibit varied phenotypes, including host adaptation and the ability to cause clinical illness in animals and humans. In the USA, Salmonella enterica serovar Kentucky is infrequently found to cause human illness, despite being the top serovar isolated from broiler chickens. Conversely, in Europe, this serovar falls in the top 10 serovars linked to human salmonellosis. Serovar Kentucky is polyphyletic and has two lineages, Kentucky-I and Kentucky-II; isolates belonging to Kentucky-I are frequently isolated from poultry in the USA, while Kentucky-II isolates tend to be associated with human illness. In this study, we analysed whole-genome sequences and associated metadata deposited in public databases between 2017 and 2021 by federal agencies to determine serovar Kentucky incidence across different animal and human sources. Of 5151 genomes, 90.3 % were from isolates that came from broilers, while 5.9 % were from humans and 3.0 % were from cattle. Kentucky-I isolates were associated with broilers, while isolates belonging to Kentucky-II and a new lineage, Kentucky-III, were more commonly associated with cattle and humans. Very few serovar Kentucky isolates were associated with turkey and swine sources. Phylogenetic analyses showed that Kentucky-III genomes were more closely related to Kentucky-I, and this was confirmed by CRISPR-typing and multilocus sequence typing (MLST). In a macrophage assay, serovar Kentucky-II isolates were able to replicate over eight times better than Kentucky-I isolates. Analysis of virulence factors showed unique patterns across these three groups, and these differences may be linked to their association with different hosts.

Identification of two distinct phylogenomic lineages and model strains for the understudied cystic fibrosis lung pathogen Burkholderia multivorans.

Burkholderia multivorans is the dominant Burkholderia pathogen recovered from lung infection in people with cystic fibrosis. However, as an understudied pathogen there are knowledge gaps in relation to its population biology, phenotypic traits and useful model strains. A phylogenomic study of B. multivorans was undertaken using a total of 283 genomes, of which 73 were sequenced and 49 phenotypically characterized as part of this study. Average nucleotide identity analysis (ANI) and phylogenetic alignment of core genes demonstrated that the B. multivorans population separated into two distinct evolutionary clades, defined as lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes). To examine the population biology of B. multivorans, a representative subgroup of 77 B. multivorans genomes (28 from the reference databases and the 49 novel short-read genome sequences) were selected based on multilocus sequence typing (MLST), isolation source and phylogenetic placement criteria. Comparative genomics was used to identify B. multivorans lineage-specific genes - ghrB_1 in lineage 1 and glnM_2 in lineage 2 - and diagnostic PCRs targeting them were successfully developed. Phenotypic analysis of 49 representative B. multivorans strains showed considerable inter-strain variance, but the majority of the isolates tested were motile and capable of biofilm formation. A striking absence of B. multivorans protease activity in vitro was observed, but no lineage-specific phenotypic differences were demonstrated. Using phylogenomic and phenotypic criteria, three model B. multivorans CF strains were identified, BCC0084 (lineage 1), BCC1272 (lineage 2a) and BCC0033 lineage 2b, and their complete genome sequences determined. B. multivorans CF strains BCC0033 and BCC0084, and the environmental reference strain, ATCC 17616, were all capable of short-term survival within a murine lung infection model. By mapping the population biology, identifying lineage-specific PCRs and model strains, we provide much needed baseline resources for future studies of B. multivorans.

High-resolution melting (HRM)-based detection of polymorphisms in the malic enzyme and glucose-6-phosphate isomerase genes for Leishmania infantum genotyping.

BACKGROUND: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites. METHODS: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples. RESULTS: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A). CONCLUSIONS: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs.

Immersion of debrided diabetic foot ulcer tissue in electrochemically generated pH neutral hypochlorous acid significantly reduces the microbial bioburden: whole-genome sequencing of Staphylococcus aureus, the most prevalent species recovered.

BACKGROUND: Diabetic foot ulcer infections (DFUIs) are the leading cause of lower-limb amputations, mediated predominantly by Staphylococcus aureus. pH-neutral electrochemically generated hypochlorous acid (anolyte) is a non-toxic, microbiocidal agent with significant potential for wound disinfection. AIMS: To investigate both the effectiveness of anolyte for microbial bioburden reduction in debrided ulcer tissues and the population of resident S. aureus. METHODS: Fifty-one debrided tissues from 30 people with type II diabetes were aliquoted by wet weight and immersed in 1- or 10-mL volumes of anolyte (200 parts per million) or saline for 3 min. Microbial loads recovered were determined in colony forming units/g (cfu/g) of tissue following aerobic, anaerobic and staphylococcal-selective culture. Bacterial species were identified and 50 S. aureus isolates from 30 tissues underwent whole-genome sequencing (WGS). FINDINGS: The ulcers were predominantly superficial, lacking signs of infection (39/51, 76.5%). Of the 42/51 saline-treated tissues yielding ≥105 cfu/g, a microbial threshold reported to impede wound-healing, only 4/42 (9.5%) were clinically diagnosed DFUIs. Microbial loads from anolyte-treated tissues were significantly lower than saline-treated tissues using 1 mL (1065-fold, 2.0 log) and 10 mL (8216-fold, 2.1 log) immersion volumes (P<0.0005). S. aureus was the predominant species recovered (44/51, 86.3%) and 50 isolates underwent WGS. All were meticillin susceptible and comprised 12 sequence types (STs), predominantly ST1, ST5 and ST15. Whole-genome multi-locus sequence typing identified three clusters of closely related isolates from 10 patients indicating inter-patient transmission. CONCLUSIONS: Short immersions of debrided ulcer tissue in anolyte significantly reduced microbial bioburden: a potential novel DFUI treatment.

Epidemiological characteristics of New Delhi Metallo-ß-Lactamase-producing Enterobacteriaceae in the Fourth hospital of Hebei Medical University.

The epidemiological characteristics of New Delhi Metallo-ß-Lactamase-Producing (NDM) Enterobacteriaceae were analyzed to provide theoretical support for clarifying the distribution characteristics of carbapenem-resistant Enterobacteriaceae (CRE) in the hospital environment and early identification of susceptible patients. From January 2017 to December 2021,42 strains of NDM-producing Enterobacteriaceae were gathered from the Fourth Hospital of Hebei Medical University, primarily Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. The micro broth dilution method combined with the Kirby-Bauer method was used to determine the minimal inhibitory concentrations (MICs) of antibiotics. The carbapenem phenotype was detected by the modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM). Carbapenem genotypes were detected by colloidal gold immunochromatography and real-time fluorescence PCR. The results of antimicrobial susceptibility testing showed that all NDM-producing Enterobacteriaceae were multiple antibiotic resistant, but the sensitivity rate to amikacin was high. Invasive surgery prior to culture, the use of excessive amounts of different antibiotics, the use of glucocorticoids, and ICU hospitalization were clinical characteristics of NDM-producing Enterobacteriaceae infection. Molecular typing of NDM-producing Escherichia coli and Klebsiella pneumoniae was carried out by Multilocus Sequence Typing (MLST), and the phylogenetic trees were constructed. Eight sequence types (STs) and two NDM variants were detected in 11 strains of Klebsiella pneumoniae, primarily ST17, and NDM-1. A total of 8 STs and 4 NDM variants were detected in 16 strains of Escherichia coli, mainly ST410, ST167, and NDM-5. For high-risk patients who have CRE infection, CRE screening should be done as soon as feasible to adopt prompt and efficient intervention measures to prevent outbreaks in the hospital.

Clinical and microbiological characteristics of bloodstream infection caused by Klebsiella pneumoniae harboring rmpA in Japanese adults.

We investigated the clinical features of bloodstream infections (BSIs) caused by Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case-control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case-control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

Effects of Aspergillus-meal prebiotic diet on the growth performance, health status and gut microbiota of Asian seabass, Lates calcarifer.

In this study, the growth performance, health status and intestinal microbiota of juvenile Asian seabass, Lates calcarifer, were assessed after dietary administration of a prebiotic product obtained from fermented Aspergillus orizae, Fermacto®. Asian seabass were fed three diets; control (without Aspergillus-meal prebiotic), 0.2% and 0.3% Aspergillus-meal prebiotic for 56 days. Fish were raised in freshwater with acceptable water quality. No significant differences were found in the growth performance and composition of dorsal fish muscle among all groups. Fish fed diets supplemented with 0.3% of Aspergillus-meal prebiotic had a significantly higher survival rate after being challenged with V. alginolyticus than fish fed with the control diet. Supplementation of the Aspergillus-meal prebiotic significantly improved immune responses by inducing higher respiratory burst, superoxide dismutase, phagocytic and lysozyme activity compared to the control group. In addition, prebiotic doses significantly induced an up-regulation of heat shock cognate 70 kDa protein (hsp70) in the liver compared to the control group. Signaling pathways were also affected with significantly higher gene expression of complement c-3 (c3), mechanistic target of rapamycin (mtor), and mammalian lethal with SEC13 protein 8 (mlst-8) in the liver of fish fed 0.3% Aspergillus prebiotic. The pro-inflammatory gene, tumor necrosis factor (tnf) and anti-inflammatory gene, transforming growth factor beta-1 (tfg-ß1) were significantly higher in the head kidney of fish offered prebiotic diets. Fish receiving Aspergillus-meal prebiotic revealed significantly higher expression of Mx gene 24 h post nervous necrosis virus injection compared to the control. Additionally, the α-diversity of gut microbiota, including genus, Pielou's evenness, Shannon diversity index, and Margalef's species richness were significantly higher in fish fed 0.3% Aspergillus-meal prebiotic than the control group. The principal component analysis eigenvector plots showed that a high abundance of beneficial bacteria, such as Entercoccus faecium, Lactococcus lactis, Macrococcus caseolyticus and Vagococcus fluvialis, along with potentially pathogenic bacteria, such as Staphylococcus sciuri and L. garvieae subsp. garvieae were present in fish treated with Aspergillus-meal prebiotic. Although dietary Aspergillus-meal prebiotic did not improve the growth performance of Asian seabass, 0.3% of Aspergillus-meal prebiotic is recommended to elevate the immunological status of fish.

Emergence of multidrug-resistant ST11 blaKPC-2 producing Klebsiella pneumoniae coharboring blaCTX-M and blaSHV in Pakistan.

INTRODUCTION: Carbapenemases are primarily responsible for the intensified spread of multidrug-resistant (MDR) K. pneumoniae by virtue of antibiotics overuse. Therefore, frequent investigation of high-risk clones especially from developing world is crucial to curtail global spread. METHODOLOGY: In this observational study, 107 K. pneumoniae were retrieved and confirmed genotypically from April 2018 to March 2020 from tertiary care hospitals in Lahore, Pakistan. Carbapenemases and extended-spectrum ß-lactamases were verified by Polymerase Chain Reaction and Sanger sequencing. Multilocus sequence typing and plasmid replicon typing were used to assign clonal lineages and plasmid replicons. RESULTS: Among the K. pneumoniae, 72.9% (78/107) strains were carbapenem resistant (CR) with 65.4% (51/78) exhibiting carbapenemase producing phenotype. Among CR K. pneumoniae 38.5% (30/78) strains exhibited the following carbapenemase genotypes: blaNDM-1 (26.7%, 8/30), blaOXA-48 (26.7%, 8/30), blaKPC-2 (20.0%, 6/30), blaVIM (10.0%, 3/30), blaNDM-1/blaOXA-48 (10.0%, 3/30), blaOXA-48/blaVIM (3.3%, 1/30) and blaOXA-48/blaIMP (3.3%, 1/30). Tigecycline and polymyxin-B retained susceptible profile. ß-lactam drugs showed intermediate to high resistance. The occurrence of CR K. pneumoniae infections was significantly associated with wound (39.7%, p = 0.0007), pus (38.5%, p = 0.009), general surgery (34.6%, p = 0.002) and intensive-care unit (26.9%, p = 0.04). blaKPC-2 producing K. pneumoniae coharboring blaCTX-M/blaSHV (66.7%) and blaCTX-M (33.3%) exhibited sequence type (ST) 258 (n = 4) and ST11 (n = 2) sequence types with IncFII, IncN, IncFIIA, IncL/M and IncFIIK plasmids. CONCLUSIONS: This is the first report describing the emergence of MDR blaKPC-2 producing K. pneumoniae ST11 coharboring blaCTX-M and blaSHV in Pakistan.

Analysis of Genetic Relatedness between Gastric and Oral Helicobacter pylori in Patients with Early Gastric Cancer Using Multilocus Sequence Typing.

The oral cavity is the second most colonized site of Helicobacter pylori after the stomach. This study aimed to compare the genetic relatedness between gastric and oral H. pylori in Japanese patients with early gastric cancer through multilocus sequence typing (MLST) analysis using eight housekeeping genes. Gastric biopsy specimens and oral samples were collected from 21 patients with a fecal antigen test positive for H. pylori. The number of H. pylori allelic profiles ranged from zero to eight since the yield of DNA was small even when the nested PCR was performed. MLST analysis revealed that only one patient had a matching oral and gastric H. pylori genotype, suggesting that different genotypes of H. pylori inhabit the oral cavity and gastric mucosa. The phylogenetic analysis showed that oral H. pylori in six patients was similar to gastric H. pylori, implying that the two strains are related but not of the same origin, and those strains may be infected on separate occasions. It is necessary to establish a culture method for oral H. pylori to elucidate whether the oral cavity acts as the source of gastric infection, as our analysis was based on a limited number of allele sequences.