Detection Of Virulence Genes Research Articles

Strain-level genomic analysis of serotype, genotype and virulence gene composition of group B streptococcus

IntroductionGBS (group B streptococcus) is an opportunistic pathogen that can colonize healthy individuals but presents significant challenges in clinical obstetrics and gynecology, as it can cause miscarriage, preterm birth, and invasive infections in newborns. To develop specific and personalized preventative strategies, a better understanding of the epidemiological characteristics and pathogenic features of GBS is essential.MethodsWe conducted a comprehensive strain-level genomic analysis of GBS, examining serotype and genotype distributions, as well as the composition and correlations of virulence genes using the blastn-short mode of the BLAST program(v2.10.0+), mlstsoftware (https://github.com/tseemann/mlst), Snippy (v4.6.0), FastTree (v2.1.11) and iTOL. The coding sequence region of virulence factors was annotated by Prodigal (v2.6.3) and Glimmer(v3.02b). We further identified host protein interacting with Srr2 by mass spectrometry analysis.ResultsWhile certain genotypes showed strong serotype consistency, there was no significant association between overall serotypes and genotypes. However, the composition of virulence genes was more closely related to the phylogeny of GBS, among which simultaneous presence of Srr2 and HygA exhibit significant association with hypervirulence. Tubulin emerged as the most distinct and abundant hit. The specific interaction of Tubulin with Srr2-BR, rather than Srr1-BR, was further confirmed by immunoblotting.DiscussionConsidering the impact of cytoskeleton rearrangement on GBS pathogenesis, this observation offers a plausible explanation for the hypervirulence triggered by Srr2. Collectively, our findings indicate that in the future clinical practice, virulence gene detection should be given more attention to achieve precise GBS surveillance and disease prevention.

Occurrence of virulence genes icaADBC and antibiotic resistance genes blaZ, mecA, and aac(6’)-Ie-aph (2’’)-Ia in coagulase-negative staphylococci isolates from neonates with sepsis at a regional referral hospital in Dar es Salaam, Tanzania

BackgroundCoagulase-negative staphylococci (CoNS) have been linked to severe conditions such as bloodstream infection, with biofilm formation regarded as the major virulence mechanism. This study determined virulence genes (VGs) (icaA, icaB, icaC, and icaD) responsible for biofilm formation and antibiotic resistance genes (ARGs) (blaZ, mecA, and aac(6’)-Ie-aph(2’’)-Ia) in CoNS isolated from blood samples of 70 neonates with sepsis in Dar es Salaam, Tanzania. We used VITEK®MS (BioMérieux, France) for speciation and phenotypically detected biofilm production using the Congo red agar. Polymerase chain reaction was performed for ARGs and VGs detection; principal component analysis was used to ascertain their relationship.ResultsOut of 70 CoNS, Staphylococcus epidermidis, 27 (36.8%) was the most commonly isolate, subsequently to Staphylococcus haemolyticus, 24 (34.3%). The intercellular adhesion genes (ica) were detected in all isolates, with IcaD found in 70 (100.0%), followed by icaB 69 (98.6%), icaC 69 (98.6%), and icaA 65 (92.9%). Four virulence gene combinations (icaADBC, icaBCD, icaACD, and icaABD) were detected, predominantly icaADBC 63 (90.0%). Sixty-one (87.1%) isolates harbored three ARGs, mostly the mecA gene 69 (98.6%), followed by blaZ 67 (95.7%) and aac (6’)-Ie-aph (2’’)-Ia, 65 (92.7%). We observed a moderate positive correlation between mecA and icaA genes (r = 0.4341). Sixty-five (97.0%) isolates resistant to penicillin harbored blaZ genes, and all 62 (100%) resistant to oxacillin harbored mecA genes. Of the 66 isolates resistant to gentamycin, 62 (93.9%) harbored the aac (6’)-Ie-aph (2’’)-Ia gene. The mecA gene contributed most of the resistance, followed by aac(6’)-Ie-aph(2’’)-Ia genes. However, the icaA and icaD genes significantly influenced the virulence of CoNS.ConclusionsMost CoNS isolated from blood samples of neonates with sepsis harbor a variety of clinically relevant virulence and resistance genes, indicating their ability to cause severe infections that are difficult to treat. Significantly, 59 (84.3%) isolates were phenotypically penicillin, gentamycin, and oxacillin resistant; the antibiotics recommended in the national guidelines for empiric treatment of neonatal sepsis.

Molecular detection of ExoU and ExoS virulence genes among clinical isolates of P. aeruginosa recovered from patients attending a private tertiary hospital in southwestern Nigeria

Molecular detection of ExoU and ExoS virulence genes among clinical isolates of P. aeruginosa recovered from patients attending a private tertiary hospital in southwestern Nigeria

Virulence and Antimicrobial Resistance Patterns of Salmonella spp. Recovered From Migratory and Captive Wild Birds.

Salmonella spp., especially those are resistant to extended-spectrum β-lactamase (ESBL), are considered as major concern to global health due to their emergence and dissemination. The aim of this study was to investigate the virulence and antimicrobial resistance (AMR) profile of Salmonella spp. from migratory and captive wild birds. A total 262 faecal samples were collected, and the identification of Salmonella spp. was carried out using a standard culture and PCR as well as molecular detection of virulence and AMR genes. The overall prevalence of Salmonella was determined to be 30.92% (95% CI = 25.63-36.75). Migratory birds exhibited highest prevalence (38.10%), whereas wild birds in captivity showed a lower prevalence (23.40%). The agfA gene was detected at a higher rate at 24.69%. Salmonella spp. exhibited 100% resistance to tetracycline, followed by 58% ampicillin and 46% streptomycin. In addition, there was a resistance rate to ceftriaxone of 17% and to colistin sulphate of 25%. Interestingly, levofloxacin alone displayed 100% sensitivity across all isolates, while ciprofloxacin and azithromycin showed 73% and 64% sensitivity, respectively. The MAR index was 0.25 and 0.42, and 74.07% of all isolates showed multidrug resistance (MDR). It was shown that migratory and captive wild birds contained ESBL genes blaTEM (94.34% and 49.06%) and blaSHV (13.33% and 10%), respectively. Genes responsible for sulphonamide (sul1) resistance were detected in 13.33% and 79% of wild and migratory birds, respectively. Salmonella has been found in captive wild and migratory birds and could act as reservoirs for the transmission of MDR and ESBL bacteria.

Antimicrobial Resistance and Pathotypes of Escherichia coli Isolates from Yellow-Legged Seagulls (Larus michahellis) in Central Italy.

Seagulls are synanthropic wild birds that can contaminate, through their droppings, beaches, urban and peri-urban environments. This concern is more serious when seagulls eliminate antimicrobial-resistant pathogenic bacteria. This study analyzed the fecal samples from 137 yellow-legged seagulls (Larus michahellis) from Central Italy. A total of 218 Escherichia coli strains were isolated and analyzed for phenotypic and genotypic antimicrobial resistance and to identify the virulence genes characterizing different pathotypes. The disk diffusion method on all isolates found relevant resistance rates to ampicillin (38.99%), tetracycline (23.85%), and enrofloxacin (21.10%). On the basis of all results obtained with this test, 62 (28.44%) isolates were classified as multidrug-resistant (MDR) and 6 (2.75%) as extensive drug-resistant (XDR). Molecular analyses conducted on the strains phenotypically resistant to carbapenems, cephalosporins, and penicillins found 9/37 (24.32%) strains positive for blaOXA-48, 52/103 (50.49%) for blaTEM, 12/103 (11.65%) for blaCMY2, 3/103 (2.91%) for blaCTX, and 1/103 (0.97%,) for blaSHV. PCR to detect virulence genes characterizing different pathotypes found that 40 (18.35%) isolates had the astA gene, indicative of the enteroaggregative (EAEC) pathotype, 2 (0.92%) had cnf1, 2 (0.92%) had cnf2, and 1 (0.46%) had cdt-IV. All five (2.29%) strains were reportable as necrotoxigenic (NTEC), while 4 (1.83%) had both eaeA and escV, reportable as enteropathogenic (EPEC). Measures to limit seagulls' access where humans and other animals reside are pivotal to reduce the risk of infection with antimicrobial-resistant and pathogenetic E. coli strains.

Suppression of virulence factors of uropathogenic Escherichia coli by Trans-resveratrol and design of nanoemulgel

BackgroundDevelopment of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized.ResultsOut of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained.ConclusionThe study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs.

Genomic Analysis and Virulence Assessment of Hypervirulent Klebsiella pneumoniae K16-ST660 in Severe Cervical Necrotizing Fasciitis

ObjectiveTo investigate the source of infection in a patient with recurrent severe neck infections caused by Klebsiella pneumoniae and to analyze the virulence of isolates obtained from different sites of the patient. MethodsWe collected preoperative neck abscess puncture fluid, intraoperative neck drainage fluid, sputum, intestinal fecal specimens, and blood samples from a patient who visited Wuxi Second People's Hospital twice between 2017 and 2018. We conducted isolation, identification, drug sensitivity tests, and string tests on the isolates. Capsule serotyping and virulence gene analysis were performed using PCR. The genetic relationship of different isolates was assessed by Multilocus Sequence Typing and virulence was evaluated using the Galleria mellonella infection model. Additionally, whole-genome sequencing was used to analyze the chromosomal and plasmid genes of one isolate. ResultsKlebsiella pneumoniae was detected in the sputum and fecal specimens from both hospitalizations, as well as the preoperative ultrasound-guided puncture fluid and intraoperative drainage fluid from the first hospitalization, resulting in six isolates. These isolates were all K16 serotype, positive in the string test, and identified as ST660 by Multilocus Sequence Typing, indicating they belonged to the same clone. Virulence gene analysis showed that wcaG, iucB, iroNB, rmpA, rmpA2, Aer, kfuBC, ureA, fimH, mrkD, uge, and peg344 were positive, while allS, cf29a, and Wzy_K1 were negative. In the Galleria mellonella virulence assay, the lethality of different isolates was dose-dependent. The K16 group showed significantly higher larval mortality compared to other control groups (including K1, K2, K5, K20, and K57 groups). Genome sequencing revealed that plasmid p17388 carried numerous virulence genes and insertion sequences, particularly ISKPN74, and showed high homology with other Klebsiella plasmids. ConclusionThis study is the first to report severe cervical necrotizing fasciitis caused by the K16-ST660 Klebsiella pneumoniae Isolate. The high virulence of these isolates was confirmed by the Galleria mellonella virulence assay and the detection of numerous virulence genes. In-depth analysis of plasmid p17388 suggests that ISKPN74 may enhance stable integration of the plasmid into the bacterial chromosome through recombinases and transposases, thereby reducing the likelihood of plasmid loss and increasing bacterial virulence. Additionally, IS5 family insertion sequences may carry extra promoters or enhancers that, when inserted upstream of mucoviscosity-associated genes such as rmpA, may increase the transcription levels of downstream genes. This ISKPN74-mediated integration or insertion reveals a complex genetic mechanism that may contribute to the severity of infections caused by ST660 isolates. Our findings offer new insights into the virulence and structure of ST660-K16 Klebsiella pneumoniae, suggesting that further investigation into the specific mechanisms by which these insertion sequences enhance virulence could aid in developing novel infection management strategies.

Occurrence of Extended-spectrum β-lactamase (ESBL) and Carbapenemase-producing Escherichia coli isolated from Childhood Diarrhoea in Yaoundé, Cameroon

IntroductionExtended-spectrum β-lactamase (ESBL)-producing pathogenic E. coli is a global public health issue, especially in sub-Saharan African countries such as Cameroon. It contributes to increase significantly hospital length of stay, morbidity, mortality and economic costs because of treatment failures. This study aims at determining the resistance background and virulence profiles of ESBL-E. coli isolates among childhood diarrhoea during the cholera outbreak occuring in Yaoundé, Cameroon.Materials and methodsDuring a four-month periods, from March 1st to June 30th, 2023, a total of 84 stool samples were collected from 90 under five children presenting clinical signs of gastroenteritis and attending four hospitals in Yaoundé, Cameroon. Bacterial identification was done using API20E and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. After extraction, genomic DNA was subjected to conventional and multiplex polymerase chain reaction methods (PCRs) for detection of resistance and virulence genes. Statistical analysis was performed using Epi info™ (7.2.5.0). Statistical significance was considered at a p-value < 0.05.ResultsOut of 150 patients contacted, 90 patients were enrolled, 84 samples were collected, 52.38%(44/84) and 3.57%(03/84) were confirmed as extended-spectrum β-lactamase and carbapenemase-producing E. coli respectively. The risk factors were analyzed, and children who drank natural fruit juice (OR: 0.4, p-value: 0.03) were found to be significantly associated with ESBL-producing E. coli. The ESBL-producing E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, colistin, and tetracycline. The blaCTX−M was more prevalent ß-lactamase resistance gene. The tetracycline resistance genes tet(A) and tet(B) were also detected. The most important virulence genes detected were FimH (81.81%) and papA (79.54%).ConclusionThese findings suggest implementing routine surveillance and screening for antimicrobial resistance among children under five. Antimicrobial stewardship strategies (ASP) need to be implemented to curb the emergence and dissemination of ESBL-producing E. coli. In addition, a national surveillance program for antimicrobial resistance needs to be implemented at local and regional levels in order to reduce morbidity in Cameroon.

Fast and Economic Microarray-Based Detection of Species-, Resistance-, and Virulence-Associated Genes in Clinical Strains of Vancomycin-Resistant Enterococci (VRE)

Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.

Molecular Properties of Virulence and Antibiotic Resistance of Pseudomonas aeruginosa Causing Clinically Critical Infections.

The increase in the number of hospital strains of hypervirulent and multidrug resistant (MDR) Pseudomonas aeruginosa is a major health problem that reduces medical treatment options and increases mortality. The molecular profiles of virulence and multidrug resistance of P. aeruginosa-associated hospital and community infections in Mexico have been poorly studied. In this study, we analyzed the different molecular profiles associated with the virulence genotypes related to multidrug resistance and the genotypes of multidrug efflux pumps (mex) in P. aeruginosa causing clinically critical infections isolated from Mexican patients with community- and hospital-acquired infections. Susceptibility to 12 antibiotics was determined using the Kirby-Bauer method. The identification of P. aeruginosa and the detection of virulence and efflux pump system genes were performed using conventional PCR. All strains isolated from patients with hospital-acquired (n = 67) and community-acquired infections (n = 57) were multidrug resistant, mainly to beta-lactams (ampicillin [96.7%], carbenicillin [98.3%], cefalotin [97.5%], and cefotaxime [87%]), quinolones (norfloxacin [78.2%]), phenicols (chloramphenicol [91.9%]), nitrofurans (nitrofurantoin [70.9%]), aminoglycosides (gentamicin [75%]), and sulfonamide/trimethoprim (96.7%). Most strains (95.5%) isolated from patients with hospital- and community-acquired infections carried the adhesion (pilA) and biofilm formation (ndvB) genes. Outer membrane proteins (oprI and oprL) were present in 100% of cases, elastases (lasA and lasB) in 100% and 98.3%, respectively, alkaline protease (apr) and alginate (algD) in 99.1% and 97.5%, respectively, and chaperone (groEL) and epoxide hydrolase (cif) in 100% and 97.5%, respectively. Overall, 99.1% of the strains isolated from patients with hospital- and community-acquired infections carried the efflux pump system genes mexB and mexY, while 98.3% of the strains carried mexF and mexZ. These findings show a wide distribution of the virulome related to the genotypic and phenotypic profiles of antibiotic resistance and the origin of the strains isolated from patients with hospital- and community-acquired infections, demonstrating that these molecular mechanisms may play an important role in high-pathogenicity infections caused by P. aeruginosa.

Detection of virulence genes in Pseudomonas fluorescens isolates from local cheese in Nineveh province

Detection of virulence genes in Pseudomonas fluorescens isolates from local cheese in Nineveh province

Bio inspired microfluidic-based analysis of Klebsiella pneumoniae virulence factors and antimicrobial resistance

The objective of this research was to examine the antibiotic resistance and microbiological traits of Klebsiella pneumoniae isolates that were responsible for liver abscesses in Qingdao, China. Between April and September 2022, isolates were taken from 120 patients at three tertiary hospitals. Rapid detection of capsule serotypes (K1, K2) and virulence genes (rmpA, aerobactin) was achieved by using microfluidic-based techniques. Testing for antimicrobial susceptibility was done with the VITEK 2 Compact system. The findings showed that there was a high frequency of extremely virulent K. pneumoniae strains, which are often linked to bacteremia and higher death rates. These strains belonged to the K1 and K2 serotypes and were primarily carrying the aerobactin and rmpA genes. The majority of isolates were antibiotic-sensitive, but the emergence of resistant strains emphasizes the necessity of continuing surveillance. The efficiency of microfluidic platforms for the quick and precise characterization of K. pneumoniae is demonstrated in this work, enabling prompt clinical interventions.

Molecular Identification and Virulence Genes Detection of Cronobacter Spp Isolated from Clinical Cases in Infants Under Two Years in Mosul City, Iraq

Cronobacter spp. are facultative-anaerobic non-spore forming Gram-negative bacteria, that are part of the Enterobacteriaceae family. They are opportunistic organisms that occasionally cause severe infections such as meningitis, necrotizing enterocolitis, and bloodstream infections in newborns and young children. There is a limited amount of information available about the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources. This study was conducted to isolate and identify Cronobacter spp from different clinical cases in children under two years in Mosul city and determine some virulence genes which are indicators for their pathogenicity. One hundred and fifty clinical specimens (diarrhea, blood and cerebrospinal fluid) were collected and cultured on Enterobacter sakazakii agar (HiCrome) and trypton soy agar. Eight Cronobacter spp were isolated and presumptively identified according to their morphological characteristics on selective media and confirmed by 16S rRNA gene sequencing and direct sequencing of ropB gene. PCR was used to amplify five potential virulence genes (cpa, hly, sip, zpx, and ompA) in order to determine their presence in our strains. The results revealed that all strains exhibited ompA and zpx, however, only four C. sakazakii strains contained cpa and hly genes. The sip gene was found in only two C. malonaticus strains. Lastly, ropB gene were detected in all Cronobacter spp isolated. Identification of Cronobacter from clinical specimens helps in understanding the sources and risks associated with these pathogens. By determining virulence genes, the study provides insights into how these bacteria cause disease which is crucial for developing targeted interventions and treatments. understanding the presence and characteristics of Cronobacter spp. in clinical cases informs diagnostic and treatment strategies, potentially leading to better patient outcomes.

Molecular Typing of Potentially Pathogenic Escherichia coli Isolated from Fresh Pasta Filata Venezuelan Cheeses

Background. Fresh pasta filata cheese is considered as one of the most important foods in the Venezuelan diet. It is typically produced by small-scale producers using raw milk. The objective of this research was to molecularly characterize the pathogenic potential of Escherichia coli strains isolated from pasta filata cheese manufactured and marketed in Venezuela. Methodology. In the period between January and March of 2019, a total of 36 strains of E. coli were isolated from a variety of pasta filata cheeses including 17 samples of mozzarella, 16 of telita, and 3 of guayanés. These strains were isolated according to the Venezuelan Commission of Industrial Standards (COVENIN) and identified by conventional methods (biochemical and phenotypic tests). Antimicrobial susceptibility was determined using the disk diffusion technique. Phylogenetic grouping and detection of virulence genes were performed by Polymerase Chain Reaction amplification. Diversity and genetic relationships were determined by Rep-PCR. Results: All strains were susceptible to the tested antibiotics. Phylogroup A (n=19) was the most frequent (52.8%), followed by groups D (n=11; 30.6%), and B1 (n=2; 5.6%). The majority of isolates carried at least two virulence genes, one coding for adhesion mechanisms (fimH) and the other for iron uptake (fyuA). Only one strain of phylogroup A presented a profile consisting of four virulence genes (fimH, fyuA, kpsMT II, and papAH). Four strains that could not be classified according to Clermont's scheme carried resistance genes as well. A heterogeneous population structure was observed by Rep-PCR of the strains. Conclusion: Results support the hypothesis that the E. coli strains isolated from the three types of pasta filata cheeses manufactured and marketed in Venezuela have identical characteristics and virulence factors to Extraintestinal Pathogenic E. coli strains observed in animals and humans, posing a potential health risk. Therefore, it is essential to improve hygienic and sanitary controls at all stages of cheese production and to implement measures for epidemiological surveillance of potentially pathogenic bacterial strains present in Venezuelan, artisanal pasta filata cheeses.

Hypermucoviscous Klebsiella Pneumoniae Caused Community-Acquired Pneumonia in Gondar, Northwest Ethiopia.

The incidence of hypermucoviscous Klebsiella pneumoniae (hmvKp), which complicates community-acquired pneumonia, has been increasing recently. This study aimed to detect hypermucoviscous K. pneumoniae and determine its antimicrobial susceptibility pattern in adult patients with community-acquired pneumonia in Northwest Ethiopia. This cross-sectional study included 39 K. pneumoniae isolates identified by using Gram stain, culture, and biochemical tests from 312 adult patients with community-acquired pneumonia at the University of Gondar Comprehensive Specialized Referral Hospital from April to June 2021. The hypermucoviscous strains were identified by using the string test. Antimicrobial susceptibility testing was performed by using the Kirby-Bauer disk dif-fusion method. Data were entered by using EPI data version 4.6 and were analyzed by using SPSS version 20. A p-value ≤ 0.05 at a 95% confidence interval was considered statistically significant. Overall, 35.9% (n = 14) of the 39 K. pneumoniae isolates were hypermucoviscous phenotype. The mean age of the hmvKp group was lower than of the cKp group (36.93 ± 12.573 vs. 53.52 ± 19.556 years, p = 0.007). All hmvKp isolates were resistant to amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole. Azithromycin resistance in the hmvKp strains was significantly higher than in the cKp group (p = 0.012). This study demonstrates that the hmvKp phenotype causes community-acquired pneumonia and a full resistance to amoxicillin-clavulanic acid and trimethoprim-sulfamethoxazole. Antimicrobial resistance was higher in the hmvKp strain than in the classic strains. Further detection of resistance genes, capsular serotypes, hypermucoviscosity-related genes, and virulence genes is necessary.

Characterizing the bacteriophage PKp-V1 as a potential treatment for ESBL-producing hypervirulent K1 Klebsiella pneumoniae ST258 isolated from veterinary specimens.

The dearth of new antibiotics necessitates alternative approaches for managing infections caused by resistant superbugs. This study aimed to evaluate the lytic potential of the purified bacteriophage PKp-V1 against extended-spectrum β-lactamase (ESBL) harboring hypervirulent Klebsiella pneumoniae (hvKp)-K1 recovered from veterinary specimens. A total of 50 samples were collected from various veterinary specimens to isolate K. pneumoniae, followed by antimicrobial susceptibility testing and molecular detection of various virulence and ESBL genes. Multilocus sequence typing of the isolates was performed to identify prevalent sequence types. The bacteriophages were isolated using the double-agar overlay method and characterized using transmission electron microscopy, spot tests, plaque assays, stability tests, and one-step growth curve assays. Among 17 (34%) confirmed K. pneumoniae isolates, 6 (35%) were hvKp, whereas 13 (76%) isolates belonging to the K1 type were positive for the wzy (K1) virulence gene. All (100%) hvKp isolates exhibited the allelic profile of ST258. Overall, PKp-V1 exhibited an 88 % (15/17; (p ≤ 0.05) host range, among which all (100 %; p ≤ 0.01) hvKp isolates were susceptible to PKp-V1. PKp-V1 exhibited a lytic phage titer of 2.4 × 108 plaque forming unit (PFU)/mL at temperatures ranging from 25°C to 37°C. The lytic phage titers of PKp-V1 at pH = 8 and 0.5% chloroform were 2.1 × 108 PFU/mL and 7.2 × 109 PFU/mL, respectively. Although the incidence of ESBL-infected K. pneumoniae in veterinary settings is worrisome, PKp-V1 phages showed considerable lytic action against the host bacterium, indicating the potential of PKp-V1 as a possible alternative therapeutic option against MDR K. pneumoniae.

Genetic Diversity, Virulence Factors and Antibiotic Resistance of Listeria monocytogenes from Food and Clinical Samples in Southern Poland.

Listeriosis is one of the most serious foodborne diseases under surveillance, with an overall mortality rate in the EU currently being high at 18.1%. Therefore, this study aims to investigate Listeria monocytogenes strains isolated from clinical and food samples for susceptibility to antimicrobials, presence of virulence factors, and genetic diversity. Species were identified using the MALDI-TOF, resistance to 11 antibiotics was determined according to EUCAST guidelines, and multiplex PCR was used for serotyping and detecting virulence genes. Strains were genotyped using the PFGE method. Clinical strains showed full sensitivity to all tested antibiotics. In total, 33.3% of strains from food products were found to be resistant to ciprofloxacin and 4.2% to tetracycline. Most of the tested isolates (79.2%) belonged to serotype 1/2a-3a, and the rest (20.8%) belonged to serotype 4ab-4b,4d-4e. Five virulence genes (prfA, hlyA, plcB, inlA, and lmo2672) were detected in all strains studied. The llsX gene was the least common, in 37.5% of clinical strains and 18.75% of strains isolated from food products. Among the analyzed strains, 13 strains displayed unique PFGE profiles. The other 11 strains belong to 3 clusters of pulsotypes: cluster 1 (2 strains), cluster 2 (6 strains), and cluster 3 (2 strains). The percentage of hospitalizations and deaths of Polish patients with listeriosis indicates the seriousness of this disease, especially in an aging society, while the molecular testing of clinical strains has been rarely performed, which makes it difficult to determine the source of infection.

Pathological and molecular characteristics of a new infectious Streptococcus agalactiae from farmed Schizopygopsis pylzovi in southern China

During the months of November and December 2022, a severe disease outbreak led to substantial mortality among Schizopygopsis pylzovi (S. pylzovi) in the Manwan reservoir of Lincang, China. The prominent clinical symptoms observed were exophthalmos with cataract, iris congestion, splenomegaly, and a pale liver. To elucidate the etiology, bacterial isolation was performed, followed by identification using Diff-Quik staining, 16s rDNA sequencing, and specific PCR. The results revealed that all three isolates were a non-hemolytic streptococcus with 93% identity to the 16S rDNA sequence of Streptococcus agalactiae (S. agalactiae), and further specific PCR demonstrated that it belonged to S. agalactiae. In addition, to further elucidate the molecular characteristics of the pathogen, a comprehensive analysis was conducted, encompassing pathological diagnosis, serotyping, multilocus sequence typing (MLST), physiological and biochemical identification, as well as the detection of related virulence genes. The findings showed that the isolates were S. agalactiae serotype Ib ST-891, exhibiting significant damage to the eye, spleen, and brain. Notably, its virulence gene profile and physiological and biochemical characteristics exhibited a significant degree of similarity to those from S. agalactiae serotype Ib ST261. Finally, experimental infection revealed that mortality rates were 100% and 75%, respectively, after exposure to 1 × 108 CFU/mL and 1 × 107 CFU/mL of this isolate suspension. It is the first report of natural infection with S. agalactiae serotype Ib ST-891 in fish, thus providing evidence for the molecular genetic diversity of fish-derived S. agalactiae.

Molecular Detection of the mecA and some Virulence Determinants in Methicillin-Resistant Staphylococcus aureus

Background: Methicillin Resistant Staphylococcus aureus (MRSA) is globally acknowledged as a prominent contributor to both hospital-acquired and community infections. Understanding key virulence factors including coagulase production, hemolysis ability and biofilm formation, is crucial. Objective: The study aimed to establish a molecular characterization of mecA gene and virulence factors genes (hla, icaA, and coa) in clinical isolates of MRSA obtained from two hospitals in Baghdad. Materials and Methods: A hundred and five isolates were obtained from clinical sources from November 2022 to March 2023 and their antibiotic sensitivity was assessed using the agar diffusion test against seven different antibiotics (Azithromycin, Ciprofloxacin, Nitrofurantoin, Rifampin, Trimethoprim, Ofloxacin and Oxacillin), through Conventional Polymerase Chain Reaction, the presence of virulence factor genes including mecA, hla, icaA, and coa, was determined in MRSA isolates. Results: All MRSA isolates (100%) harbored the mecA, hla, and icaA genes while the coa gene was recognized in 50% of the isolates. Regarding antibiotic susceptibility, all MRSA isolates (100%) demonstrated sensitivity to Nitrofurantoin. Additionally, 96.8% of the isolates were sensitive to Oxacillin. Conclusion: Molecular detection of methicillin resistance genes and virulence genes can be used to diagnose MRSA isolates in hospitals. The presence of these genes may affect their pattern of sensitivity to antibiotics.

Immune-histochemistry and molecular characterization techniques for detection of Pasteurella spp virulence genes in cattle

Immune-histochemistry and molecular characterization techniques for detection of Pasteurella spp virulence genes in cattle