Detection Of Virulence Factors Research Articles

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings

BackgroundColistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT).ResultsThis study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 μg/mL), while broth microdilution identified 48 (MIC = 32–128 μg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357).ConclusionThe global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

A Metagenomic Investigation of Potential Health Risks and Element Cycling Functions of Bacteria and Viruses in Wastewater Treatment Plants.

The concentration of viruses in sewage sludge is significantly higher (10-1000-fold) than that found in natural environments, posing a potential risk for human and animal health. However, the composition of these viruses and their role in the transfer of pathogenic factors, as well as their role in the carbon, nitrogen, and phosphorus cycles remain poorly understood. In this study, we employed a shotgun metagenomic approach to investigate the pathogenic bacteria and viral composition and function in two wastewater treatment plants located on a campus. Our analysis revealed the presence of 1334 amplicon sequence variants (ASVs) across six sludge samples, with 242 ASVs (41.22% of total reads) identified as pathogenic bacteria. Arcobacter was found to be the most dominant pathogen accounting for 6.79% of total reads. The virome analysis identified 613 viral genera with Aorunvirus being the most abundant genus at 41.85%. Approximately 0.66% of these viruses were associated with human and animal diseases. More than 60% of the virome consisted of lytic phages. Host prediction analysis revealed that the phages primarily infected Lactobacillus (37.11%), Streptococcus (21.11%), and Staphylococcus (7.11%). Furthermore, our investigation revealed an abundance of auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and phosphorus cycling within the virome. We also detected a total of 113 antibiotic resistance genes (ARGs), covering major classes of antibiotics across all samples analyzed. Additionally, our findings indicated the presence of virulence factors including the clpP gene accounting for approximately 4.78%, along with toxin genes such as the RecT gene representing approximately 73.48% of all detected virulence factors and toxin genes among all samples analyzed. This study expands our understanding regarding both pathogenic bacteria and viruses present within sewage sludge while providing valuable insights into their ecological functions.

Phenotypic and genotypic characterization of virulence markers and antifungal susceptibility of oral Candida species from diabetic and non-diabetic hemodialysis patients

BackgroundPatients with chronic kidney disease undergoing hemodialysis are often colonized by Candida species with high possibility of fungal infections. The purposes of this study were to determine the prevalence of Candida species, evaluate antifungal susceptibility profile, biofilm formation, proteinase and phospholipase activities, and the frequency of virulence genes in the Candida species isolated from the oral mucosa of hemodialysis diabetic (DM) and non-diabetic (non-DM) patients.MethodsThis study identified several species of Candida isolated from 69 DM and 58 non-DM patients on hemodialysis using phenotypic methods and PCR–RFLP technique. The identification of C. albicans and C. glabrata complex was performed by HWP1 gene and four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f), respectively. Antifungal susceptibility to amphotericin B, fluconazole, itraconazole, voriconazole, and caspofungin was assessed according to CLSI M27-A3/S4. The biomass, metabolic activity of biofilm, proteinase (Prz), phospholipase (Pz), and molecular study for virulence genes were assessed using crystal violet, XTT assay, agar-based hydrolytic enzyme, and PCR technique, respectively.ResultsCandida prevalence was 44.9% with 47.8% and 41.4% among DM and non-DM patients, respectively (P = .045). The species identified were C. albicans (49.5%), C. glabrata (16.5%), C. tropicalis (12%), C. kefyr (8.8%), C. parapsilosis (6.6%), C. dubliniensis (3.3%), and C. lusitaniae (3.3%). The antifungal susceptibility profile showed that all Candida isolates were sensitive to amphotericin B, itraconazole, voriconazole, and caspofungin whereas fluconazole resistance was observed in 6.3% (MIC ≥ 64 μg/mL) of C. albicans and 6.6% of C. glabrata (MIC ≥ 64 μg/mL). The susceptible- dose-dependent rate was found in 10.5% of C. albicans. The Prz values of C. albicans ranged from 0.37 to 0.66 for the DM and 0.44–0.73 for the non-DM group (P < 0.05). The non-albicans Candida (NAC) species produced higher degree of biomass and metabolic activity compared to C. albicans (P < 0.05). Furthermore, significant (p < 0.05) correlations were detected between the biofilm formation with Prz values and fluconazole MICs. The most detected virulence factors were ALS3 and Sap5.ConclusionsThese results showed the importance of prevalence of NAC species in hemodialysis patients. Investigating antifungal susceptibility profile made a better understanding of the role of virulence markers in the pathogenesis of Candida strains.

Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex).

Lactococcosis, caused by members of the genus Lactococcus, represents a devastating disease inducing mass mortalities and economic losses in many fish species worldwide. The present work aimed to compare the whole genome sequences of three different serotypes of Lactococcus garvieae isolated from diseased cultured striped jack (Pseudocaranx dentex) in Ehime prefecture, Japan. The three serotypes showed different virulence in the challenge test using Japanese amberjack (Seriola quinqueradiata). The genome sequencing revealed that two of the strains (serotype I and serotype III) were identified as L. garvieae, while the third strain (serotype II) was identified as L. formosensis. The chromosome sizes of the three serotypes ranged from 1.9 to 2.0 Mb; the GC content ranges were 38.2 to 38.9%; and the numbers of predicted protein-coding sequences (CDSs) were from 1922 to 1959. Only the serotype II harbours two plasmids, sizes of around 14 kb and 9 kb. The detected virulence factors varied among the different serotypes with some shared factors like adherence, anti-phagocytosis, secretion system, toxin (haemolysin), serum resistance, antimicrobial resistance and others. The genomes also contained factors responsible for resistance to toxic compounds. The genome of the serotype III tended to encode more prophage regions than the other serotypes.

Extensively drug-resistant Acinetobacter baumannii: role of conjugative plasmids in transferring resistance.

Acinetobacter baumannii is one of the most successful pathogens that can cause difficult-to-treat nosocomial infections. Outbreaks and infections caused by multi-drug resistant A. baumannii are prevalent worldwide, with only a few antibiotics are currently available for treatments. Plasmids represent an ideal vehicle for acquiring and transferring resistance genes in A. baumannii. Five extensively drug-resistant A. baumannii clinical isolates from three major Jordanian hospitals were fully sequenced. Whole-Genome Sequences (WGS) were used to study the antimicrobial resistance and virulence genes, sequence types, and phylogenetic relationship of the isolates. Plasmids were characterized In-silico, followed by conjugation, and plasmid curing experiments. Eight plasmids were recovered; resistance plasmids carrying either aminoglycosides or sulfonamide genes were detected. Chromosomal resistance genes included blaOXA-66, blaOXA-91, and blaOXA-23, and the detected virulence factors were involved in biofilm formation, adhesion, and many other mechanisms. Conjugation and plasmid curing experiments resulted in the transfer or loss of several resistance phenotypes. Plasmid profiling along with phylogenetic analyses revealed high similarities between two A. baumannii isolates recovered from two different intensive care units (ICU). The high similarities between the isolates of the study, especially the two ICU isolates, suggest that there is a common A. baumannii strain prevailing in different ICU wards in Jordanian hospitals. Three resistance genes were plasmid-borne, and the transfer of the resistance phenotype emphasizes the role and importance of conjugative plasmids in spreading resistance among A. baumannii clinical strains.

Assessment of virulence factors and antimicrobial resistance among the Pseudomonas aeruginosa strains isolated from animal meat and carcass samples.

Pseudomonas aeruginosa bacteria are emerging causes of food spoilage and foodborne diseases. Raw meat of animal species may consider a reservoir of P. aeruginosa strains. The present survey was done to assess the prevalence, antibiotic resistance properties and distribution of virulence factors among the P. aeruginosa strains isolated from raw meat and carcass surface swab samples of animal species. Five hundred and fifty raw meat and carcass surface swab samples were collected from cattle and sheep species referred to as slaughterhouses. P. aeruginosa bacteria were identified using culture and biochemical tests. The pattern of antibiotic resistance was determined by disk diffusion. The distribution of virulence and antibiotic resistance genes was determined using polymerase chain reaction. Forty-seven of 550 (8.54%) examined samples were contaminated with P. aeruginosa. The prevalence of P. aeruginosa in raw meat and carcass surface swab samples were 6.57 and 12%, respectively. P. aeruginosa isolates showed the maximum resistance rate toward penicillin (87.23%), ampicillin (85.10%), tetracycline (85.10%), gentamicin (65.95%) and trimethoprim (57.44%). The most commonly detected antibiotic resistance genes were BlaCTX-M (53.19%), blaDHA (42.55%) and blaTEM (27.65%). The most commonly detected virulence factors was ExoS (42.55%), algD (31.91%), lasA (31.91%), plcH (31.91%) and exoU (25.53%). Meat and carcass surface swab samples may be sources of resistant and virulent P. aeruginosa, which pose a hygienic threat in their consumption. However, further investigations are required to identify additional epidemiological features of P. aeruginosa in meat and carcass surface samples.

Study of Virulence Factor Production in Candida Species Colonizer Isolated from Diabetic Patients

Candida is a yeast like fungus. It causes candidiasis which is a spectrum of disease from cutaneous, mucosal, systemic &amp; multisystem dissemination. It is a coloniser of mucocutaneous surfaces of body but it is an opportunistic fungus causing severe type of infection. Objective of the study was to detect colonization of Candida species in diabetic patient &amp; to detect virulence factors i.e., phospholipase, proteinase, esterase activity &amp; homolysis activity in isolated strains of Candida from colonised diabetic patients. Throat swab and urine sample were collected from diabetic patients visiting the General Medicine OPD/Ward diagnosed with diabetes mellitus before six months and was submitted in microbiology department. Candida species were identified tested for production of virulence factors. Total 30 diabetes mellitus patients diagnosed 6 months before. In 41-65 years age groups, Candida colonization was found predominantly, 63% patients. Amongst these 78% had fasting sugar levels greater than the normal limits. The study revealed a strong correlation between higher fasting blood sugar levels and Candida colonization. The predominant isolate was Candida albicans in throat followed by Candida glabrata. Out of 27 Candida species isolates, 16 strains showed production of virulence factors. Amongst these 44 % (7/16) strains were positive for proteinase production, 38 % (6/16) were positive for esterase production, 13% (2/16) strains were positive for hemolysin and single strain (6%, 10/16) showed the production of phospholipase. The study concludes that Candida albicans was the predominant colonising species found followed by Candida glabrata in diabetic patients. Non-Candida albicans species can be seen as an emerging colonizing species in the diabetic patients and thereby are increasingly gaining clinical importance. Colonized or commensal Candida species showed in vitro production of virulence factor.

Perillaldehyde mitigates virulence factors and biofilm formation of Pseudomonas aeruginosa clinical isolates, by acting on the quorum sensing mechanism in vitro.

The incidence of biofilm linked catheter-associated urinary tract infections is increasing worldwide and Pseudomonas aeruginosa is one of the major causes. Perillaldehyde (PLD): as a natural, widely used flavouring agent, has been reported to possess various pharmacological properties. We hypothesized that PLD can inhibit biofilm formation and virulence factor (VF) production by P. aeruginosa by hampering the quorum sensing (QS) system(s). Minimum inhibitory concentration (MIC) of PLD was assessed for standard strain and two multi-drug resistant catheter isolates of P. aeruginosa utilizing the microdilution method. Microtiter plate assay, crystal violet staining and scanning electron microscopy were used to evaluate the biofilm inhibition property. CFU was utilized to assess the antifouling property of PLD. Detection of VFs and expression analysis of virulence determinants were applied to investigate the anti-virulence activity. Gene expression and molecular docking studies were also executed to explore the QS inhibition and binding of PLD with QS receptors. In the present study, PLD has significantly inhibited biofilm formation and antivirulence activity at sub-MIC levels (2.5 and 3.5 mM) in all the tested strains. In addition, molecular docking studies revealed a significant affinity towards QS receptors. Perillaldehyde, being a non-toxic food flavouring agent, significantly inhibited biofilm formation and exhibited antifouling property. PLD exhibited significantly reduced levels of VFs (p < 0.001) and their respective genetic determinants (p < 0.001). Gene expression analysis and molecular docking studies confirmed the interactions of PLD to the QS receptors, indicating the plausible mechanism for the anti-virulence property. This study identified the anti-virulence potential of PLD and provided mechanistic insights. PLD can be a suitable, non-toxic candidate for countering biofilms and associated pathogens, contributing to the prevention of biofilm-associated nosocomial infections.

Case of necrotic enteritis associated with campylobacteriosis and coccidiosis in an adult Indian peacock (Pavo cristatus)

BackgroundTo date, Campylobacter jejuni has not been found to be pathogenic to peafowl. The available publications show that out of a total of 44 samples tested from peafowl, this bacterium was isolated only in two cases. Eimeria pavonina infestations in the peafowl have been described, but no fatal cases have been reported yet.Case presentationThe four-year-old peacock was presented with chronic diarrhea, emaciation and weakness. Post mortem examination revealed enlarged and pale kidneys, small intestinal mucosal necrosis and thickening of intestinal wall, and pericardial effusion. The histopathological examination revealed necrotic enteritis with marked mononuclear cells infiltration associated with the presence of coccidia, additionally there was histological evidence of septicemia in liver and kidneys. Bacteria identification was based on light microscopy of the small intestine sample, culture, and biochemical tests. Further identification was based on PCR. Antimicrobial susceptibility profile was created by determination of minimal inhibitory concentration (MIC) values for 6 antimicrobial agents from 5 different classes. PCR assays were performed to detect virulence factors genes responsible for motility, cytolethal distending toxin production, adhesion and internalization. Bacteriology of the small intestine sample showed abundant growth almost exclusively of Campylobacter jejuni, resistant to ciprofloxacin, gentamycin and ampicillin. Bacteria was sensitive to Amoxicillin + clavulanic acid, tetracycline, and erythromycin. All tested virulence factors genes have been detected. The parasitological examination was performed by microscopic examination of fresh faeces and intestinal content, and revealed the moderate number of Eimeria pavonina, Histomonas meleagridis, single Capillaria spp. eggs as well Heterakis spp. like parasites.ConclusionThe above case shows that a virulent isolate of Campylobacter jejuni in combination with a parasitic invasion may cause chronic enteritis in peafowl, which most likely led to extreme exhaustion of the host organism and death.

Molecular characterization and antimicrobial resistance of Enterococcus faecalis isolated from seafood samples.

Background Enterococcus faecalis is considered an opportunistic foodborne pathogen. The present study aimed to assess the prevalence, antimicrobial resistance, virulence characters, and molecular typing of E. faecalis strains isolated from seafood samples.MethodsTwo hundred and seventy‐six seafood samples were collected. E. faecalis was isolated from samples using bacterial culture. Furthermore, the disk diffusion assessed their antimicrobial resistance. Also, the distribution of virulence factors was determined using polymerase chain reaction (PCR) assay. Random amplified polymorphic DNA (RAPD) method was used for their molecular typing.ResultsFifty‐six of 276 (20.2%) seafood samples were contaminated with E. faecalis. Fish harboured the highest contamination rate (30.0%). Isolates harboured the highest resistance rate towards oxacillin (100%), tetracycline (100%), erythromycin (100%), cefoxitin (89.2%), cefazolin (87.5%), trimethoprim‐sulfamethoxazole (85.7%), rifampin (69.6%), clindamycin (69.6%), and gentamicin (64.2%) antimicrobials. Efa (100%), ebpA (89.2%), ebpB (58.9%), ebpC (53.5%), and esp (51.7%) were the most commonly detected virulence factors among E. faecalis isolates. RAPD–PCR analysis showed 11 different molecular clusters considering the closeness of more than 80%.ConclusionSeafood samples were considered reservoirs of virulence and resistant E. faecalis strains. Different molecular clusters of isolates may reflect their diverse sources of contamination.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a “One-Health” Perspective

Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Virulence Factors and Prevalence of Udder´s Pathogens in Dairy Cows during the Peripartal Period

Data presented in this study described the prevalence of udder pathogens and presence of virulence factors in staphylococci isolated from mastitis in dairy cows. The practical part of study was realized in five different dairy herds of Slovak spotted cattle breed located in Eastern Slovakia with conventional (non-organic) farming. At the beginning of lactation (during first two months after calving) were comprehensively investigated 960 cows from monitored herds. The comprehensive examination included clinical examination, sensory analysis of milk from fore stripping of each udder quarter, followed by assessment of the California mastitis test (CMT) and laboratory analyses of bacteria pathogens. Screening the health status of the mammary gland revealed that 314 cows (32.7%) had positive CMT score (1-3) for one or more quarters. Out of 230 infected milk samples, representing 24.0% of all dairy cows examined, were the most commonly isolated staphylococci (59.1% of positive findings), followed by E. coli (11.3%), streptococci Str. uberis (9.1%), Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of coagulase negative stafylococci (98 isolates) and S. aureus (38 isolates) were detected some virulence factors such as production of hemolysins (lysines ß and δ), gelatinase, biofilm, and hydrolyze of DNA. Isolated S. aureus, S. chromogenes and S. warneri had the most numerous representation of detected virulence factors, as demonstrated by the increased incidence of clinical forms of mastitis compared to less virulent strains.

Characterization of Enterococcus spp. isolated from a fish farming environment in southern Brazil.

The aim of present study is to characterize the resistance and virulence profile of enterococci isolated from aquaculture excavated ponds and masonry tanks (6 samples) in southern Brazil. Samples were cultured in selective medium, 10 colonies were randomly selected from each sample, which were identified by MALDI-TOF and tested against 13 antimicrobials. The presence of resistance (tetL, tetM, tetS, ermB and msrC) and virulence (ace, esp, agg, cylA and gelE) genes were determined by PCR. A total of 79 enterococci were identified, and Entecococcus faecalis (44.3%) and E. casseliflavus (36.7%) were the most prevalent species isolated. Sixty-five strains (82.3%) were resistant to at least one of the antimicrobials tested, whereas 27 (34.2%) strains were multiresistant. The overall percentages of antimicrobial resistant isolates were: 58.2% to rifampicin, 40.5% to fluoroquinolones, 36.7% to erythromycin and 30.4% to tetracycline. The tetL and tetM genes were found in 57.7% of the tetracycline-resistant strains; and msrC in 31.01% of erythromycin-resistant strains. The most frequently detected virulence factors were ace and gelE genes. Although limited to a single farm, these data suggest that aquaculture may be a reservoir of resistant and virulent enterococci. This study is the first step towards enhancing our understandingof distribution, resistance and virulence profile in enterococci isolated from fish farming environments in the south Brazil.

Phenotypic and genetic analysis of virulence factors in multidrug- sensitive and multidrug-resistant clinical isolates of Pseudomonas aeruginosa

This study aimed to correlate the pattern of antimicrobial susceptibility, phenotypic production of virulence factors, the occurrence of virulence factors genes and the clonal profile of clinical isolates of Pseudomonas aeruginosa of a tertiary hospital in Recife-PE. The 30 clinical isolates (15 multidrug-sensitive (MDS) and 15 multidrug-resistant (MDR)) were analyzed using phenotypic methods to detect virulence factors (alkaline protease, hemolysin, phospholipase C, lipase, and pigments). The detection of the aprA, lasA, lasB, plcH, and toxA genes was performed through specific PCRs, and the clonal profile was assessed using ERIC-PCR. The results revealed cephalosporins being the class eliciting the highest percentage of resistance; the MDR isolates were all resistant. Among the MDS isolates, all were sensitive to carbapenems and quinolones. The MDR isolates produced less virulence factors such as pyocyanin and lipase, and exhibited lower expression of toxA and lasA genes, whereas the MDS isolates produced less hemolysin and phospholipase C. There was no difference between the groups for alkaline protease production and aprA gene expression. All the isolates produced pyocyanin and expressed lasB and plcH genes. A great genetic diversity was found, and it was possible to observe 28 genetic profiles. Clones were present among the MDR isolates. The occurrence of virulence factors in almost all the isolates studied suggests their high level of pathogenicity, demonstrating that this pathogen is capable of accumulating numerous virulence factors, and in some cases, is associated with multidrug resistance, which makes it difficult to treat these infections.

Virulence factors and antibiotic resistance properties of Streptococcus species isolated from hospital cockroaches.

Hospital cockroaches are probable sources of pathogenic bacteria. The present investigation was performed to assess the antibiotic resistance properties and distribution of virulence factors in the Streptococcus spp. isolated from hospital cockroaches. Six hundred and sixty cockroach samples were collected. Cockroaches were washed with normal saline, and the achieved saline was used for bacterial culture. Isolated Streptococcus spp. were subjected to disk diffusion. The distribution of virulence factors and antibiotic resistance genes were assessed using a polymerase chain reaction. The prevalence of S. pyogenes, S. agalactiae, and S. pneumonia amongst examined samples was 4.82%, 1.66%, and 6.96%, respectively. Cfb (53.93%), cyl (52.8%), scaa (51.68%) and glna (50.56%) were the most commonly detected virulence factors. Pbp2b (71.91%), pbp2x (58.42%), mefA (46.06%), ermB (46.06%) and tetM (46.06%) were the most commonly detected antibiotic resistance genes. Streptococcal spp. harbored the highest prevalence of resistance against tetracycline (80.89%), trimethoprim (65.16%), and penicillin (56.17%). To the best of our knowledge, this is the first prevalence report of virulence factors and antibiotic resistance genes in the Streptococcal spp. isolated from American, German, and oriental hospital cockroaches in Iran. Our findings indicated a certain role for cockroaches in nosocomial pathogens transmission in the hospital environment.

Bacterial protease activity as a biomarker to assess the risk of non-healing in chronic wounds: Results from a multicentre randomised controlled clinical trial.

Millions worldwide suffer from chronic wounds challenging clinicians and burdening healthcare systems. Bacteria impede wound healing; however, the diagnosis of excessive bacterial burden or infection is elusive. Clinical signs and symptoms of infection are inaccurate and unreliable. This trial evaluated a novel, point-of-care, lateral flow diagnostic designed to detect virulence factors released by the most common bacteria found in chronic wounds. A multicentre prospective cohort clinical trial examined the efficacy of a diagnostic test in detecting bacterial proteases taken from swab samples of chronic venous, arterial, pressure and mixed aetiology chronic wounds. Two hundred and sixty six wounds were included in the analysis of the study. The wounds were tested at the start of the study after which investigators were permitted to use whatever dressings they desired for the next 12 weeks. Healing status at 12 weeks was assessed. The presence of elevated bacterial protease activity decreased the probability of wound healing at 12 weeks. In contrast, a greater proportion of wounds were healed at 12 weeks if they had little or no bacterial protease activity at study start. In addition, the presence of elevated bacterial protease activity increased the time it takes for a wound to heal and increased the risk that a wound would not heal, when compared to the absence of bacterial protease activity. The results of this clinical trial indicate that bacterial protease activity, as detected by this novel diagnostic test, is a valid clinical marker for chronicity in wounds. The diagnostic test offers a tool for clinicians to detect clinically significant bacteria in real time and manage bacteria load before the clinical signs and symptoms of infection are evident.

GENOTYPIC DETECTION OF THE VIRULENCE FACTORS OF UROPATHOGENIC ESCHERICHIA COLI (UPEC) STRAINS ISOLATED FROM PREGNANT FEMALES AND THEIR CORRELATION WITH ANTIBIOTIC RESISTANCE PATTERN

Urinary Tract Infections (UTIs) during pregnancy are among the most common infections worldwide and can lead to poor perinatal and maternal outcomes, especially in developing countries and requires medical treatment as soon as it is detected. Virulent and resistant strains of Escherichia coli are the most important causative agents for UTIs. The present investigation aimed to detect virulence factors of Uropathogenic Escherichia coli (UPEC), including pap, fim, sfa, aer and hly genes in isolates collected from symptomatic and asymptomatic pregnant females by multiplex polymerase chain reaction (PCR) assay and determine their correlation with antibiotic resistance patterns. Two hundred urine samples collected from pregnant females with or without symptoms of UTI were admitted to antenatal clinic at Obstetrics and Gynecology Department of Al- Zahraa University Hospital in Cairo. Out of 200 cultured urine samples, the cultures that showed ≥ 105 CFU/ml were identified to have significant growth. Found significant bacteriuria was detected in 61 samples and these samples selected for further analysis in this study, the overall prevalence of UTI among pregnant females was (30.5%). Symptomatic pregnant females with UTI were (48.5%) more than asymptomatic (11.3%). The most frequently isolated species was S. saprophyticus (35.0%),followed by E. coli (26.2%),S .aureus (19.4%), C .albicans (5.8%),S. epidermidis (2.9%) and each of K. pneumonia, K .oxytoca, P .vulgaris, E.fecalis, Bacillus spp were(1.9%),while P. auroginosa had the least percentage of isolation (1.0%). Antimicrobial susceptibility pattern was done for 27 UPECisolates showed the highest level of resistance (100%) against β-lactams as (Ampicillin, Penicillin and Ceftriaxone), nitrofurantoin, and ampicillin/sulbactam, while the lowest level of resistance (66.7%) against Cotrimoxazole. Multiple drug resistance (resistance to two or more drugs) was observed in (100%) of the UPEC isolates. The higher level of resistance to antibiotics was observed in symptomatic bacteriuria more than asymptomatic. Among two predominant uropathogens, E. coli showed complete resistance up to 5 different antibiotics of total 11 antibiotics (45.5%) while S. saprophyticus showed complete resistance (18.2%), so E. coli showed higher virulence and resistance than S. saprophyticus to cause UTI, so subjected to Multiplex PCR to detect its virulence factors, which included pap, fim, sfa, aer and hly genes Theadhesive fimH gene was the most frequent in UPEC isolates (92.6%) . These virulence genes detected from total UPEC isolates of symptomatic cases were higher than asymptomatic. On the other hand, UPEC strains carrying the virulence genes were more resistant to the antibiotics used, so these observations confirm the important role of virulence genes of UPEC in in existence of symptoms of UTI and the drug resistance.

Occurrence of Escherichia coli in salmon sashimis commercialized in restaurants from Londrina - PR

The population interest for fish consumption has increased, mainly due to several beneficial nutritional properties presented by this food. In this context, oriental culinary also brings different eating habits as consume raw food, such as sashimi. A relevant food contaminant of fecal origin is Escherichia coli, able to become potentially harmful, when it acquires virulence factors, as Shiga toxin-producing E. coli (STEC). This study aimed to evaluate 30 samples of salmon sashimi regarding the presence of E. coli, as well as perform the genotypic characterization of virulence factors associated with STEC. Three samples were collected from 10 different restaurants, specialized in Japanese culinary in the city of Londrina - PR. The E. coli identification was performed using the Colilert® chromogenic substrate technique and biochemical tests, and for the investigation of virulence genes, stx1 and stx2, the polymerase chain reaction (PCR) was used. Among the 30 samples analyzed, 15 (50%) presented contamination by E. coli. However, in no sample were detected virulence factors associated with STEC. Although human diseases associated with STEC are poorly described in Brazil, it is possible to verify that fish, mainly those consumed raw, are potential transmitters of E. coli to humans. This can compromise the food safety of these products and, thus, characterize them as unsuitable for consumption. Therefore, it is necessary the adoption of preventive measures of contamination by E. coli in products intended to human consumption, beyond more research that can verify the potential of STEC as a fish contaminant.

Characteristics of Vibrio vulnificus isolates from clinical and environmental sources

Researchers have developed multiple methods to characterize clinical and environmental strains of Vibrio vulnificus. The aim of our study was to use four assays to detect virulence factors in strains from infected patients and those from surface waters/sediments/oysters of South Carolina and the Gulf of Mexico. Vibrio vulnificus strains from clinical (n = 81) and environmental (n = 171) sources were tested using three real-time PCR methods designed to detect polymorphisms in the 16S rRNA, vcg and pilF genes and a phenotypic method, the ability to ferment D-mannitol. Although none of the tests correctly categorized all isolates, the differentiation between clinical and environmental isolates was similar for the pilF, vcgC/E and 16S rRNA assays, with sensitivities of 74.1–79.2% and specificities of 77.4–82.7%. The pilF and vcgC/E assays are comparable in efficacy to the widely used 16S rRNA method, while the D-mannitol fermentation test is less discriminatory (sensitivity = 77.8%, specificity = 61.4%). Overall percent agreement for the D-mannitol fermentation method was also lower (66.7%) than overall percent agreement for the 3 molecular assays (78.0%–80.2%). This study demonstrated, using a large, diverse group of Vibrio vulnificus isolates, that three assays could be used to distinguish most clinical vs environmental isolates; however, additional assays are needed to increase accuracy.

Seroepidemiology, Antimicrobial Susceptibility and Virulence Characteristics of Clinical Klebsiella pneumoniae Isolates in Mansoura University Hospitals

Background: Klebsiella pneumoniae is one of the crucial causes of nosocomial and community-acquired infections that can result in various infections in human. Objectives: The present study aim to investigate the prevalence of capsular serotypes, antimicrobial susceptibility and virulence characteristics of K. pneumoniae isolated from different Mansoura University Hospitals. Methodology: K. pneumoniae isolates were collected from different clinical sources at Mansoura University Hospitals. The antimicrobial susceptibility to 14 different antibiotics was determined by disk diffusion method. The capsular serotypes were assessed by quelling test. Serum resistance, haemagglutination, biofilm, lipase, protease and lecithinase enzymes production were assessed phenotypically. Moreover, four virulence genes (rmpA, fimH, kfuBC and wabG) were detected by PCR. The genetic relatedness among isolates was investigated using ERIC-PCR molecular typing. Results: Seventy-three isolates were confirmed as K. pneumoniae. The vast majority of isolates demonstrated MDR patterns (72.6%) including a high resistance rate to the beta-lactam antibiotics (ampicillin: 98.6%, amoxicillin-clavulanic acid: 97.26 %, piperacillin: 97.26 %, amoxicillin: 93.15% and cefotaxime: 94.52%). K1 and K2 were the main serotypes found among the isolates, K1 serotype was the predominant (79.45%). It was found that serum resistance was the highest detected virulence factor among isolates (95.9%) and lipase was the lowest detected factor (19.2%). Haemagglutination was detected in 63% of the isolates especially from rectal swab (83.3%) and sputum (72.7%). The biofilm formation was detected mainly among urine and blood isolates. rmpA, fimH, kfuBC and wabG genes were harbored by 20.5%, 92%, 66% and 94.5% of isolates, respectively. ERIC- PCR showed high genetic diversity (100%, typability, Simpson’s index of diversity= 1). Conclusion: The current study revealed the high antibiotic resistance levels, pathogenic potential, and genetic diversity among K. pneumoniae isolated from different clinical sources which is considered a serious health problem that necessitates interventions to control its spread.