Virulence Markers Research Articles

Physiological race characterisation of Fusarium oxysporum strains infecting tomato employing candidate pathogenicity genes and host resistance.

Physiological races of 14 strains of Fusarium oxysporum f.sp. lycopersici were established by PCR profiling SIX gene expressions. No amplification of the SIX4 (Avr1) gene was observed in any of the 14 strains. Based on amplification of the SIX3 (Avr2) gene, 6 strains were distinguished as race 2. Race 2 strains are known to contain identical SIX3 sequences and differ from race 3 strains by single point mutations. Hence, based on polymorphic amplicons of the SIX3 gene detected by stringent PCR conditions, 8 strains were identified as race 3. The identity of the physiological races of the strains was validated by inoculating on three germplasm lines, EC-814916, FEB-2 and Pusa Rohini carrying I-2, I-3 and no I gene, respectively. The race 2 and race 3 strains were avirulent on EC-814916 and FEB-2 lines, respectively. All the 14 fungal strains were pathogenic on Pusa Rohini, the Fusarium wilt susceptible cultivar lacking R genes and exhibited different levels of virulence. In evaluating two other potential pathogenicity genes, Fow1 and Fow2 as markers for virulence, their expressions were observed among both the races of the Fol strains, and hence are not potential candidates for physiological race discrimination. However, strong expressions of the genes in the root tissues inoculated with the highly virulent strain, TOFU-IHBT in comparison to the uninoculated control indicated their roles in fungal pathogenicity. To understand the role of these pathogenicity genes in countering the host defence mechanisms, their expressions in response to ROS and phenolics, the earliest known defence mechanisms of host plants were assessed. In H2O2, the Fow2 gene expressed 1.4-fold greater than that of the control. On the contrary, in relation to the control, the expressions of Fow1 were strongly repressed exhibiting 0.7-to 0.8-fold lesser at 0.1 mM through 3 mM concentrations than that of the control indicating that the gene is modulated by the phenolic acid indicating the roles of Fow2 and Fow1 in alleviating oxidative stress and targeted by the phenolic acid, respectively.

Phenotypic ESBL and non-phenotypic ESBL isolates of Klebsiella pneumoniae exhibit differing responses to induced antimicrobials resistance and subsequent antibiotic cross-resistance.

To investigate the effect of adapting Klebsiella pneumoniae clinical isolates harboring ESBL genes to cetrimide (CT) in terms of subsequent cross-resistance to other biocides and antibiotics, and to investigate changes to virulence markers, such as biofilm formation and efflux activity. The changes between phenotypic extended spectrum β-lactamases (pESBL) expressing isolates and non-phenotypic ESBL (npESBL) isolates was compared. Kl. pneumoniae isolates (14 pESBL and 17 npESBL) were adapted to increasing concentrations of CT, until 4 × MIC was reached. The MIC of the adapted isolates was tested against chloroxylenol (CX) and chlorhexidine. Disk diffusion techniques were used to determine the susceptibility of the isolates to different antibiotics. Biofilm formation was assessed for the isolates using the crystal violet method and efflux pump activity was studied using the ethidium bromide assay. After CT adaptation, 100% of npESBL isolates and 85.7% of pESBL isolates showed increase in CT MIC after CT adaptation. While 41.2% of npESBL and 57.1% of the pESBL isolates showed a cross-resistance with chlorhexidine. CT adaptation resulted in a significant decrease in the susceptibility of npESBL isolates to aztreonam and cefotaxime compared to pESBL isolates, which could be linked to the increase in efflux activity of npESBL compared to pESBL. Biofilm formation was significantly increased after CT adaptation regardless of the type of isolate. The extensive use of biocides in the environment can induce cross-resistance to other biocides and antibiotics, and can increase the ability of bacteria to form biofilms. The response of bacteria to biocide adaptation differs between pESBL and npESBL isolates, although the effect is strain specific.

Detection of putative virulence traits in Aeromonas spp. from brazilian food chain through phenotypic tests and polymerase chain reaction

Aeromonads are natural inhabitants of aquatic environments and may be associated with numerous infections in humans and animals. The human disease may range from self-limiting diarrhea to a more severe form. The pathogenesis of infections are multifactorial, because of their wide variety of virulence factors. This study aimed to evaluate virulence markers in Aeromonas isolates and determinate their virulence profiles. There were analyzed 120 strains of A. caviae (n = 57) and A. hydrophila (n = 63) from human, animal and environmental sources between 2008 and 2012. All isolates were examined to detect extracellular virulence enzymes by phenotypic activity and the presence of virulence genes hlyA, aerA, lip, gcat, ser, act, alt and exu by PCR. We observed more than 90% of positivity for at least one phenotypic virulence factors and all of them had at least two of the virulence genes measured. Among the virulence enzymes detected, the DNase was present in 93.33% of the isolates and hemolytic activity was detected in 62.5%. Collagenase and elastase were found in 13.33% and 10.83% of the strains, respectively. We found exu and gcat in 100% of the isolates, lip in 40.83%, aerA in 40.83%, hlyA in 40%, alt in 19.16%, act in 17.5% and ser in 11.66%. It was possible to observe different combinations of virulence factors between the isolates showing the multifactorial virulence among the isolates. The diversity of virulence profiles found in this study hint heterogeneity in clones circulating in our environment.

A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics.

Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml-1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.

A Comprehensive Genomic Analysis of the Emergent Klebsiella pneumoniae ST16 Lineage: Virulence, Antimicrobial Resistance and a Comparison with the Clinically Relevant ST11 Strain.

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health.

Molecular analysis of enteropathogenic Escherichia coli (EPEC) isolates from healthy food-producing animals and humans with diarrhoea.

Enteropathogenic Escherichia coli (EPEC) is a pathogen associated with acute diarrhoea in humans. To determine whether EPEC isolated from healthy food-producing animals possesses the same virulence gene repertoire as EPEC isolated from human with diarrhoea, we compared six typical EPEC (tEPEC) and 20 atypical EPEC (aEPEC) from humans with diarrhoea and 42 aEPEC from healthy animals (swine, sheep and buffaloes), using pulsed-field gel electrophoresis (PFGE), virulence markers, serotyping and subtyping of eae and tir genes. We found that human and animal isolates shared virulence genes, including nleB, nleE and nleF, which were associated with human diarrhoea. Serogroups and serotypes identified in isolates of food-producing animals such as O26:H11, O128:H2, O76:H7, O103, O108, O111 and O145, have previously been implicated in human disease. The subtypes eae and tir were also shared between human and animal isolates, being eae-γ1 and eae-β1 the most prevalent in both groups, while the most common tir subtypes were α and β. Despite PFGE analysis demonstrating that EPEC strains are heterogeneous and there was no prevalent clone identified, EPEC isolated from humans and food-producing animals shared some characteristics, such as virulence genes associated with human diarrhoea, indicating that food-producing animals could play a role as reservoirs for those genes.

Molecular Characterization Of Non-Fermenting Gram-Negative Bacilli And Its Virulence Markers From Various Clinical Samples In A Tertiary Care Center, South India

Introduction: Non-fermentative Gram-negative bacilli (NFGNB) are an important cause of healthcare-associated infections, and theyare primarily opportunistic pathogens.Aim and objectives: To isolate and characterize the non-fermenting Gram-negative bacilli (NFGNB) from various clinical samples ina tertiary care center. To detect the virulence markers and drug resistance using molecular methods.Material and methods: Non-fermentative Gram-negative bacilli (NFGNB) from various clinical samples were identified using standardbacteriological identification methods. Conventional PCR for detection of MBLs genes of P. aeruginosa such as blaIMP, blaVIM, blaNDMand a virulence marker, Exo S gene and also MBL genes of A. baumannii such as blaIMP, blaVIM, blaNDM, blaOXA-23, blaOXA-58, and avirulence marker, OmpA gene.Results: Out of 104 NFGNB isolates Pseudomonas aeruginosa was the most common bacteria comprising 62% (n=65), followed byAcinetobacter baumannii 25% (n=26), Acinetobacter lwoffii 4% (n=4), Pseudomonas flourescens 3% (n=3), Burkholderia cepacia 3%(n=3), Stenotrophomonas maltophilia 1% (n=1), Myroides odoratimimus 1% (n=1), and Rhizobium radiobacter 1% (n=1). The resultof P. aeruginosa showed that the blaVIM gene was expressed in 25 %(n=2), the blaNDM gene was expressed in 50 %(n=4) and blaIMPwas found in 25 %(n=2). For A. baumannii, the blaNDM gene was expressed in 33% (n=6), blaOXA-23 gene was expressed in 78%(n=14), blaOXA-58 gene was expressed in 56% (n=10) with no expression of blaIMP and blaVIM genes.Conclusion: In our study we report the increased prevalence of MBL genes in P. aeruginosa and A. baumannii. This highlights theimportance of accurate identification of MBL producers and judicious use of carbapenems. Early identification and continuedsurveillance of these multidrug resistant organisms will help to prevent their spread in a hospital environment.

Review of the Zoonotic Importance of Salmonellosis and Associated Risk Factors

Salmonella is one of the most common and significant human and animal food-borne pathogens that cause salmonellosis, which has high medical and monetary costs. Salmonella has a big impact on public health because it can spread zoonotic and foodborne diseases to people. Many domestic and wild animals intestinal tracts serve as the main reservoir for Salmonella, which can result in the direct or indirect contamination of a variety of foods with fecal organisms. Salmonellae are etiological agents of diarrheal and systemic infections. They can be shed in large numbers on the faces of clinical cases and carrier animals, causing contamination of the environment and frequently resulting in subclinical infections. Infection in food animals often leads to contamination of meat, eggs, milk and cheese. The organism can also spread through direct contact with infected humans, animals, or feces contaminated environments. Infected food handlers may also act as a source of contamination for foodstuffs, because of the increasing antibiotic resistance of organisms and companion animals, animals are an important source of Salmonella infection for humans. Salmonella can colonize its hosts by invading, adhering, and bypassing the host’s intestinal defense mechanisms, such as gastric acid, thanks to a variety of virulence markers and determinants, including flagella, capsule, plasmids, adhesion systems, and secretion systems. Diagnosis is based on the isolation of the organism either from tissues collected aseptically at necropsy or from faces, rectal swabs or environmental samples, food products, and feedstuffs; prior or current infection of animals by some agents may also be diagnosed serologically.

Virulomic Analysis of Multidrug-Resistant Klebsiella pneumoniae Isolates and Experimental Virulence Model Using Danio rerio (Zebrafish).

This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored blaKPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types.

Analysis of the Virulence and Inflammatory Markers Elicited by Enteroaggregative Escherichia coli Isolated from Clinical and Non-Clinical Sources in an Experimental Infection Model, India

Enteroaggregative Escherichia coli (EAEC) is highly heterogeneous in virulence; we wanted to understand the pathogenic potential of EAEC isolated from various clinical and non-clinical sources in an animal model. We infected male BALB/c mice in six mice/groups with 50 EAEC isolates isolated from clinical and non-clinical sources. We studied colonization, weight loss, stool shedding, and inflammatory markers and their relationship with 21 virulence genes and phylogroups, EAEC organ burden, and histopathological changes. We detected significantly more inflammatory changes and fecal lactoferrin and calprotectin levels in mice infected with EAEC isolated from symptomatic cases. In clinical EAEC isolates, the presence of chromosomal genes (aap (46%), aaiC (23.3%), SPATEs (pet (13.3%), sat (20%), sigA, and pic (6.6%)), the adhesive variantsof EAEC (agg4A (53.3%), aggA (53.3%), aafA (36.6%), andagg3A (40%)), and the master regulator gene aggR (66.6%) were associated with higher levels of lactoferrin and calprotectin. Additionally, 70% (9/13) of EAEC isolated from acute diarrheal cases bearing chuA (70%) in our study were assigned to groups B2 (4 isolates) and D (5 isolates). Real-time PCR analysis revealed that colonization by EAEC strains from different clinical and non-clinical sources occurs up to 10–15 days of life. Even from non-diarrheal stools and non-clinical sources, EAEC strainshad the potential to cause prolonged colonization, weight loss, and inflammation in the intestine, though the degree varied. Moreover, a better understanding of EAEC pathogenic pathways is desperately needed in different clinical scenarios.

Phage controlling method against novel freshwater-derived Vibrio parahaemolyticus in ready-to-eat crayfish (Procambarus clarkii)

Vibrio parahaemolyticus is a halophilic foodborne pathogen majorly isolated from seafood, threatening public health worldwide. However, our recent study reported the presence of this bacterium in freshwater crayfish, which were rarely identified and investigated. Its pathogenicity and genomic features remain unclear, and the controlling method to inhibit this bacterium in ready-to-eat (RTE) crayfish is not developed. Compared with a clinical strain (ATCC17802), the representative strain (LVP1) from freshwater crayfish showed higher pathogenicity in a zebrafish model, indicating its hypervirulence and foodborne infection risk. Unlike most clinical V. parahaemolyticus isolates that carried tdh and (or) trh, two classic virulence factor genes associated with clinical infections, the hypervirulent LVP1 lacked these two genes, indicating it is a novel strain and other unknown virulence factors play key roles in its pathogenicity. Genomic and phylogenetic analyses demonstrated this strain is V. parahaemolyticus, while it is phylogenetically distant from other global isolates. Therefore, LVP1 is considered a novel V. parahaemolyticus strain from freshwater crayfish, being hypervirulent, and lacking virulence marker genes. The antimicrobial resistant genes drfA6 and qnrV1 were unique in LVP1 and absent in other reference V. parahaemolyticus strains. Antimicrobial susceptibility testing confirms it as multidrug resistance, with resistance to ampicillin, amoxicillin/clavulanate, trimethoprim, and colistin. To control this novel and multidrug-resistant V. parahaemolyticus strain in food production chains, we developed a phage cocktail and applied it to the surface of RTE crayfish meat, resulting in a significant decrease in the bacterial load by 2.36 log10 CFU/mL. These data highlight freshwater food products pose threats to public health through ‘farm-to-fork’ transmission of hypervirulent V. parahaemolyticus and reveal the phage cocktail as a promising method to attenuate this bacterium in RTE food, emphasizing the necessity to prevent food contamination caused by this bacterium from freshwater products.

Integrative Pathogenicity Assay and Operational Taxonomy-Based Detection of New Forma Specialis of Fusarium oxysporum Causing Datepalm Wilt.

Pathogenicity-associated genes are highly host-specific and contribute to host-specific virulence. We tailored the traditional Koch’s postulates with integrative omics by hypothesizing that the effector genes associated with host-pathogenicity are determinant markers for virulence, and developed Integrative Pathogenicity (IP) postulates for authenticated pathogenicity testing in plants. To set the criteria, we experimented on datepalm (Phoenix dactylifera) for the vascular wilt pathogen and confirmed the pathogen based on secreted in xylem genes (effectors genes) using genomic and transcriptomic approaches, and found it a reliable solution when pathogenicity is in question. The genic regions ITS, TEF1-α, and RPBII of Fusarium isolates were examined by phylogenetic analysis to unveil the validated operational taxonomy at the species level. The hierarchical tree generated through phylogenetic analysis declared the fungal pathogen as Fusarium oxysporum. Moreover, the Fusarium isolates were investigated at the subspecies level by probing the IGS, TEF1-α, and Pgx4 genic regions to detect the forma specialis of F. oxysporum that causes wilt in datepalm. The phylogram revealed a new forma specialis in F. oxysporum that causes vascular wilt in datepalm.

Prevalence and distribution of pathogenic genes in Campylobacter jejuni isolated from poultry and human sources.

Campylobacter jejuni is one of the most common bacterial causes of human gastroenteritis. Despite its public health importance, the virulence factors and mechanisms underlying C. jejuni pathogenesis remain poorly understood and the relationships between these genes and the sources of the strains are not clear. We aimed to determine the virulence profiles of C. jejuni isolated from poultry and human cases of Campylobacteriosis. A total of 50 strains of C. jejuni isolated from poultry and human cases of Campylobacteriosis were screened by polymerase chain reaction (PCR) for the presence of six pathogenic genes (flaA, iam, wlaN, cdtA, cdtB, cdtC). A total of 40% (10/25) of the human isolates presented only one virulence marker. In contrast, 64% (16/25) of the poultry-derived strains showed four or five virulence markers. cdtA and flaA occurred more frequently in poultry-derived strains than in human strains. Ten different virulence profiles were observed among the human isolates and 11 among the poultry strains. Only four profiles were common to both sources: profiles 3 (flaA, cdtA, cdtB, and cdtC), 5 (cdtA and cdtB), 7 (flaA and cdtB), and 10 (iam, flaA, cdtA, cdtB, and cdtC). The human-derived strains had a higher Shannon diversity index (1.9396) and Simpson index (0.8367), indicating a more diversified population than found in poultry (1.7742 and 0.7333, respectively). We found variations in the genetic profiles of the circulating strains based on the isolation source and genes that are potentially pathogenic to humans were detected in poultry-derived strains.

Clostridium perfringens associated with dairy farm systems show diverse genotypes

Clostridium perfringens is a bacterial species of importance to both public and animal health. Frequently found in food system environments, it presents a risk to food animal health such as dairy herds, and may cross contaminate associated ingredients or food products, with potential to cause sporadic and outbreaks of disease in human populations, including gastroenteric illness. In this study, we characterized C. perfringens isolated from bovine, caprine, and ovine dairy farm systems (n = 8, 11 and 4, respectively). Isolates were phenotypically screened for antimicrobial sensitivity profiling, and subjected to whole genome sequencing to elucidate related genetic markers, as well as examine virulence gene markers, mobile genetic elements, and other features. Both toxin type A and type D isolates were identified (78 % and 22 % of isolates, respectively), including 20 novel sequence types. Resistance to clindamycin was most prevalent among antibiotics screened (30 %), followed by erythromycin (13 %), then penicillin and tetracycline (4 %), although an additional 3 isolates were non-susceptible to tetracycline. Most isolates harboured plasmids, which mobilised virulence markers such as etx, cpb2, and resistance markers tetA(P), tetB(P), and erm(Q), on conjugative plasmids. The presence of type D isolates on caprine farms emphasizes the need for control efforts to prevent infection and potential enterotoxemia. Clostridium perfringens enterotoxin (cpe) was not identified, suggesting lower risk of gastrointestinal illness from contaminated foods, the presence of other virulence and antimicrobial resistance markers suggests farm hygiene remains an important consideration to help ensure food safety of associated dairy foods produced.

Shiga Toxin Subtypes, Serogroups, Phylogroups, RAPD Genotypic Diversity, and Select Virulence Markers of Shiga-Toxigenic Escherichia coli Strains from Goats in Mid-Atlantic US.

Understanding Shiga toxin subtypes in E. coli from reservoir hosts may give insight into their significance as human pathogens. The data also serve as an epidemiological tool for source tracking. We characterized Shiga toxin subtypes in 491 goat E. coli isolates (STEC) from the mid-Atlantic US region (stx1 = 278, stx2 = 213, and stx1/stx2 = 95). Their serogroups, phylogroups, M13RAPD genotypes, eae (intimin), and hly (hemolysin) genes were also evaluated. STEC-positive for stx1 harbored Stx1c (79%), stx1a (21%), and stx a/c (4%). Those positive for Stx2 harbored stx2a (55%) and Stx2b (32%), while stx2a/stx2d and stx2a/stx2b were each 2%. Among the 343 STEC that were serogrouped, 46% (n = 158) belonged to O8, 20% (n = 67) to 076, 12% (n = 42) to O91, 5% (n = 17) to O5, and 5% (n = 18) to O26. Less than 5% belonged to O78, O87, O146, and O103. The hly and eae genes were detected in 48% and 14% of STEC, respectively. Most belonged to phylogroup B1 (73%), followed by D (10%), E (8%), A (4%), B2 (4%), and F (1%). M13RAPD genotyping revealed clonality of 091, O5, O87, O103, and O78 but higher diversity in the O8, O76, and O26 serogroups. These results indicate goat STEC belonged to important non-O157 STEC serogroups, were genomically diverse, and harbored Shiga toxin subtypes associated with severe human disease.

Molecular Characterization of Extended Spectrum β-Lactamase (ESBL) and Virulence Gene-Factors in Uropathogenic Escherichia coli (UPEC) in Children in Duhok City, Kurdistan Region, Iraq.

Background: The presence of extended-spectrum β-lactamase (ESBL)-producing bacteria among uropathogens is significantly increasing in children all over the world. Thus, this research was conducted to investigate the prevalence of E. coli and their antimicrobial susceptibility pattern, and both genes of ESBL-producing E. coli resistant and virulence factor in UTIs patients among children in Duhok Province, Kurdistan, Iraq. Method: a total of 67 E. coli were identified from 260 urine samples of pediatric patients diagnosed with UTIs aged (0–15 years) which were collected from Heevi Pediatric Teaching Hospital, from August 2021 to the end of February 2022. Result: a high proportion of UPEC infections at ages <5 years and the rates among girls (88%) were significantly higher than those among the boys. A wide variety of E. coli are resistant to most antibiotics, such as Amoxicillin, Ampicillin and Tetracycline, and 64% of them were positive for ESBL. Interestingly, the presence of both the ESBL marker genes (blaTEM, and blaCTX-M) as well as both virulence marker genes (pai and hly) were detected in above 90% of E. coli. Conclusion: the data illustrate an alarming increase in UPEC with ESBL production and the emergence of multidrug-resistant drugs in the early age of children. The public health sectors should further monitor the guidelines of using antibiotics in Kurdistan, Iraq.

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ​+ ​M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ​+ ​M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.

ПАТОГЕННЫЙ ПОТЕНЦИАЛ БАКТЕРИЙ ГРУППЫ ESKAPE, ВЫДЕЛЕННЫХ ИЗ РАН: ХАРАКТЕРИСТИКА ФЕНОИ ГЕНОТИПИЧЕСКИХ МАРКЕРОВ И ВОЗМОЖНОСТЬ ИХ ПРАКТИЧЕСКОГО ПРИМЕНЕНИЯ

Backgroud. Evaluation of the biological properties of isolates obtained from the clinical samples of patients will increase the informative value of the microbiological study and optimize the interpretation of the culture results in order to substantiate further wound treatment tactics. Aim. To analyze the phenotypic and genotypic properties of ESKAPE bacteria and to determine the possibility of practical use of the parameters characterizing the bioprofile of the isolate when interpreting the results of microbiological culture of the wound swabs. Material and methods. Pheno- and genotypic markers of the pathogenic potential of ESKAPE isolates were analyzed: Enterococcus faecalis (n=93), Staphylococcus aureus (n=177), Klebsiella pneumoniae (n=7), Acinetobacter baumannii (n=32), Pseudomonas aeruginosa (n=45), Enterobacterales (Proteus mirabilis, n=26). Microorganisms were isolated from the wound swabs of patients with acute and chronic wounds, who were admitted to the burn department of the Gomel City Clinical Hospital No. 1 for specialized medical care during the period 2012-2020. Integumentary tissue defects were represented by post-traumatic wounds (mechanical trauma, thermal burns), postnecrotic wounds (after purulent-inflammatory diseases of the skin and underlying tissues), trophic ulcers (on the stumps of the lower extremities after prolonged wearing of the prosthesis; neurotrophic ulcers after mechanical trauma to peripheral nerves); pressure ulcers stage III after prolonged tissue compression. When describing the isolates, the duration of the wounds and the presence of clinical signs of infectious inflammation were taken into account. Results. The relationship between the clinical state of the wound (no signs of inflammation, critical colonization, infection) and the bioprofile of the isolate obtained from the wound swabs was determined. Potentially pathogenic S. aureus, possessing colonization, invasive and cytotoxic properties, was found in wounds with a minimum duration of up to 4 days, but the absence of clinical manifestations of infection suggested contamination. Biofilm formation, persistent properties, genetic markers of pathogenicity in S. aureus and E. faecalis in combination with pathological changes in fine-grained granulations in the wound indicated colonization. The inflammatory status of the wounds (critical colonization and infection according to NERDS&amp;STONEES criteria) from which P. aeruginosa and A. baumannii were isolated, the presence of genetic virulence markers, and antibiotic resistance indicated the clinical significance of these isolates as etiological agents of the infectious process. P. mirabilis isolated from critically colonized and infected wounds did not have the full range of virulence markers in all cases, especially in associations. K. pneumoniae was isolated only from the wounds showing signs of inflammation and demonstrated various combinations of genetic determinants of virulence, variability in capsular polysaccharide overproduction and biofilm formation. Conclusion. An algorithm has been developed for interpreting the results of microbiological culture of wound swabs with the use of pheno- and genotypic markers, which are recommended for determining the leading pathogen that colonizes the wound and disrupts the healing process. The algorithm allows assessing the etiological significance of mixed cultures isolated from critically colonized and infected wounds, which will determine the further tactics of treating patients.

Virulence genes and antibiotic resistance profile of Vibrio species isolated from fish in Egypt.

Vibrio species are significant pathogens affecting aquatic species. Around 12 species of Vibrio can cause a gastrointestinal illness (gastroenteritis) in humans resulting from eating contaminated food such as raw or undercooked shellfish. The indiscriminate use of antibiotics accelerates the development of resistance representing a severe challenge for controlling Vibrio outbreaks. In this study, the antibiotic resistance profile and the prevalence of pathogenic Vibrio species of apparently healthy and diseased fishes isolated from different types of fish in Kafr EL-Sheikh Governorate in Egypt during 2018 were determined. Samples obtained from fishes were inoculated onto a Vibrio-selective medium (TCBS) and phenotypically identified using the biochemical characteristics and representative cultures were checked by PCR to confirm the identified isolates. In the present study, V. alginolyticus (16.00%) was the predominant species followed by V. cholerae (7.33%) and V. parahaemolyticus (5.33%). The tested isolates were resistant to ampicillin (80.00%) and sensitive to ciprofloxacin and norfloxacin (100%). A total number of 15 Vibrio isolates (five Vibrio parahaemolyticus, five V. alginolyticus, and five V. cholerae) were screened for five housekeeping genes and pathogenic virulence markers by PCR. Results showed that 100% of the V. parahaemolyticus isolates carried the tlh gene and 60.00% carried the tdh gene. In V. alginolyticus, 100% of the isolates carried the collagenase gene 0.00% carry the tdh gene; and 80.00% of V. cholerae isolates carried the ctx gene. The results showed that many isolates in this study had virulence characteristics that might correspond with the potential of infections and diseases.

Correlation of phenotypic and genotypic virulence markers, antimicrobial susceptibility pattern, and outcome of Pseudomonas aeruginosa sepsis infection.

Pseudomonas aeruginosa (PA) possesses several virulence genes that enable them to evade the immune system and to cause injury in the host tissue. However, the number of studies that characterized the virulence genes profile in PA sepsis is limited. The main objective of this study was to identify and characterize virulence genes in PA causing sepsis, as well as investigate the relationship between virulence genes, antimicrobial susceptibility patterns, and infection outcomes. A prospective study, conducted between October 2020-October 2021, isolates were recovered from blood samples and identified using standard microbiological procedures. Phenotypic techniques were used to screen for capsule, siderophore production, biofilm formation, serum resistance, hemolysin production, and protease. Molecular techniques were performed to screen for alginate D (alg D), exoenzyme S (Exo S), exotoxin A (tox A), phospholipase H (plc H), phospholipase N (plc N), and elastase B (las B). Kirby-Bauer disc diffusion method was used to determine the antimicrobial susceptibility pattern of isolates, which was then interpreted according to the CLSI 2021 guidelines. Out of the n=215 Gram-negative bacteria recovered from sepsis patients during our study, n=20 were Pseudomonas aeruginosa. PA isolates were susceptible to all antibiotics tested except for 3 of the isolates that were resistant to gentamycin, 2 to imipenem, and 1 to ceftazidime, cefepime, meropenem, tobramycin, and amikacin. The most prevalent virulence genes present were capsule (100%), siderophore production (100%), alg D (100%), Las B (100%), and Tox A (100%). Our study found that PA causing sepsis harbours a high level of virulence genes. However, the high presence of virulence factors was not statistically associated with antimicrobial susceptibility, as most isolates in our study were susceptible to the antibiotics tested.