Detection Of Virulence Genes Research Articles

Molecular detection of virulence genes and Extended Spectrum Beta-lactam producing enzymes of Proteus mirabilis isolates from fishes in Federal Capital Territory (FCT) Abuja, Nigeria

Molecular detection of virulence genes and Extended Spectrum Beta-lactam producing enzymes of Proteus mirabilis isolates from fishes in Federal Capital Territory (FCT) Abuja, Nigeria

Molecular Analysis of Enterococcus Faecalis Isolates in a 4-year Period.

In the present research, we aimed to determine the characteristics of E. faecalis strains collected from an Iranian Children's Hospital for four years. Sixty-seven E. faecalis isolates with virulence genes detection, variable-number tandem repeat (VNTR), and multiple-locus variable-number tandem repeat analysis (MLVA) typing were investigated. A high frequency of virulence genes belonged to gelatinase (73%) and Enterococcus faecalis (62%). The MLVA of 67 E. faecalis isolates revealed 52 VNTR patterns and 38 MLVA types (MTs). Furthermore, genetic diversities with the majority of the MT1 as a major MT in different Wards of the Children's Hospital were found.

Analysis of Microbiological Quality and Antibiotic Resistance Patterns in Milk Supply Chain

The widespread consumption of milk for its nutritional value and health benefits brings the risk of milk-borne diseases due to the presence of various microorganisms, including antibiotic-resistant pathogens. This has led to an increased focus on ensuring the safety of milk products across the supply chain by dairy industries. The study aimed to evaluate microbiological parameters and detect multi-antibiotic-resistant pathogens at three specific supply points and to explore the association between the presence of residual antibiotics and the resistant isolates in milk samples. About 50 milk samples, including raw, soon-after-processed, packaged marketed pasteurized, and UHT milk, were subjected to microbiological analysis. This involved assessing the total bacterial count (TBC) and total coliform count (TCC), conducting antibiotic susceptibility tests through disk and well diffusion assays, detecting virulence genes in multi-antibiotic resistant isolates using gene-specific PCR, and analyzing residual antibiotics by HPLC. The study revealed that the quality of raw milk samples was unacceptable (TBC >4.5x107 CFU/mL and TCC >5.6x104 CFU/mL), while pasteurized samples from processing plants had lower counts than those from retail stores (TBC >5x105 CFU/mL and TCC >1.6x104 CFU/mL) indicating post-pasteurization contamination. About 70.37% of the isolates were Gram-negative, with Escherichia coli (21.4%) and Vibrio (18.8%) being the most prevalent. Resistance to antibiotics was substantial, particularly against ampicillin (86.3%), tetracycline (76%), and ciprofloxacin (58.9%). Gene-specific PCR analysis detected uidA, oprL, and oprI virulence genes in multi-drug-resistant Escherichia coli and Pseudomonas sp. respectively. The study also revealed a direct association between the presence of residual antibiotics and the resistant isolates, emphasizing the need for dairy industry improvements. As high bacterial counts in milk can pose health risks by fostering antibiotic-resistant pathogens, it is essential to mitigate microbiological contamination in the milk supply chain through the implementation of various precautionary measures.
 Bioresearch Commu. 10(1): 1462-1473, 2024 (January)

Impact and Diversity of ESBL-Producing Klebsiella pneumoniae Recovered from Raw Chicken Meat Samples in Türkiye.

The interrelationship between human, animal and environmental sectors leads to the spread of antibiotic resistance due to selective pressures, evolutionary traits and genomic evolution. In particular, the frequent use of antibiotics in livestock inevitably influences the emergence of specific resistance determinants in human strains, associated with reduced treatment options in clinical therapy. In this study, ESBL-producing Klebsiella pneumoniae strains isolated from chicken meat samples were evaluated for public health implications in Türkiye. Whole-genome sequencing was used for genetic dissection and phylogenetic comparison of their genomes. The isolates were assigned to four MLST types (ST147, ST37, ST2747 and ST219); two of them were found to represent the ST147 clone associated with severe human infections worldwide. In addition to cephalosporins, high resistance levels to quinolones/fluoroquinolones were identified phenotypically, caused by acquired resistance genes and chromosomal point variations. One isolate was also found to carry the qacE∆1 efflux transporter gene, which confers tolerance to quaternary ammonium compounds. The detection of virulence genes (i.e., that coding for enterobactin) associated with the pathogenicity of K. pneumoniae suggests a public health impact. Thus, comprehensive information on the occurrence and impact of K. pneumoniae from livestock is needed to derive appropriate management strategies for consumer protection. In this study, it was shown that poultry meat serves as a reservoir of clinically emerging multidrug-resistant high-risk clones.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment

BackgroundColistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments.ResultsFour hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 μg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia.ConclusionOur findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Study of the Genomic Characterization of Antibiotic-Resistant Escherichia Coli Isolated From Iraqi Patients with Urinary Tract Infections.

Urinary tract infection is one of the last diseases prevalent in humans, with various causative agents affecting 250 million people annually, This study analyzed UTIs in Iraqi patients caused by Escherichia coli. ESBL enzymes contribute to antibiotic resistance. The research aimed to analyze ESBL gene frequency, resistance patterns, and genetic diversity of E. coli strains; Between Dec 2020 and May 2021, 200 urine samples were collected, cultured on blood agar, EMB, and MacConkey's plates, samples incubated at 37°C for 24h. Positive samples (> 100cfu/ml) underwent Kirby-Bauer and CLSI antibiotic susceptibility testing. PCR detected virulence genes, Beta-lactamase coding genes, and biofilm-associated resistance genes in E. coli isolates; Out of 200 isolates, 80% comprised Gram-positive and Gram-negative bacteria. Specifically, 120 isolates (60%) were Gram-negative, while 40 isolates (20%) were Gram-positive. Among Gram-negative isolates, 20% were identified as E. coli. Remarkably, all E. coli strains showed resistance to all tested antibiotics, ranging from 80 to 95% resistance. The E. coli isolates harbored three identified resistance genes: blaTEM, blaSHV, and blaCTXM. Regarding biofilm production, 10% showed no formation, 12% weak formation, 62% moderate formation, and 16% strong formation; our study found that pathogenic E. coli caused 20% of UTIs. The majority of studied E. coli strains from UTI patients carried the identified virulence genes, which are vital for infection development and persistence.

The Molecular Detection and Antimicrobial Profiles of Selected Bacterial Pathogens in Slaughterhouses in Riyadh City, Saudi Arabia

Inadequate hygienic conditions and poor handling are the primary causes of contamination in abattoirs. This study aimed to identify and molecularly detect pathogenic bacteria in sheep meat at slaughterhouses in Riyadh City, Saudi Arabia. Additionally, the study evaluated the sensitivity of these bacteria to various antimicrobials. In total, 150 samples were collected three times every two months from November 2021 to March 2022 from four abattoirs located in the south, west, east, and north of Riyadh. Pathogenic bacteria were separated using selective media, and the Vitek 2 system was utilized to identify all species and test their response to antibiotics. PCR was employed to detect virulence genes. The four pathogenic bacteria identified in all samples were Escherichia coli (12%), Klebsiella pneumoniae (9.3%), Salmonella enterica (7.3%), and Pseudomonas aeruginosa (6.6%). Abattoir D had a high number of bacteria isolated in January. K. pneumoniae and S. enterica exhibited resistance to ampicillin. S. enterica also demonstrated resistance to gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole. P. aeruginosa was resistant to tigecycline. PCR results indicated positive tests for the E. coli gene FimH, the P. aeruginosa genes plcH and toxA, and the K. pneumoniae gene mrkD. Pathogenic bacteria with positive results for virulence genes have the potential to cause contamination and human diseases. To improve quality and reduce contamination, the government must address the issue of providing adequate and safe water for activities in all abattoirs in Riyadh City.

MOLECULAR IDENTIFICATION OF VIRULENCE PROPERTIES AND VIRULENCEASSOCIATED GENE (ISS) OF E. COLI ISOLATED FROM BROILER CHICKS

The present study identified virulence genes for existence of virulence genes increased the serum survival (Iss) for determining the pathogenicity regarding E. coli in broiler chicks. The results indicated that 45 (36.7%) of the 120 samples collected from dead and morbid broiler chicks that have been infected with E. coli, and the difference between non-pathogenic and pathogenic was investigated. Hemolysin production, motility, and the agglutination test have been also shown to be positive. In vitro tests revealed that three isolates have been classed as moderately, highly, and slightly virulent, based on their pathogenicity features. Broiler chicks have been typified by the monovalent antisera had indicated that (31.74%) have been identified as E. coli serotype O2. Slightly and highly pathogenic isolates have been responsive to chloramphenicol, penicillin, gentamicin, and streptomycin, while moderately pathogenic isolates have been only susceptible to the gentamicin and chloramphenicol. The use of the PCR as a valid approach for detecting virulence genes resulted in an increase in serum survival (Iss) in 15 (65.2%) of the total sample

Detection of virulence genes of Staphylococcus aureus isolated from raw beef for retail sale in the markets of Ulaanbaatar city, Mongolia

BackgroundStaphylococcus aureus (S. aureus) is a highly virulent pathogen that causes food-borne illness, food poisoning, skin and soft tissue infections, abscesses, mastitis, and bacteremia. It is common for meat and meat products to become contaminated with S. aureus due to dirty hands, food storage conditions, food production processes, and unhygienic conditions, causing food poisoning. Therefore, we aimed to isolate S. aureus strain from the raw beef and reveal virulence genes and antibiotic resistance profile from isolated S. aureus strains.MethodsIn this study, 100 samples of raw beef were collected from 4 major market stalls in Ulaanbaatar city, Mongolia. S. aureus was detected according to the ISO 6888–1:2021 standard, and the nucA gene encoding the species-specific thermonuclease was amplified and confirmed by polymerase chain reaction (PCR). In the strains of S. aureus isolated from the samples, the genes encoding the virulence factors including sea, sed, tsst, eta, etb, and mecA were amplified by multiplex PCR. These genes are encoded staphylococcal enterotoxin A, enterotoxin D, toxic shock syndrome toxin, exotoxin A, exotoxin B and penicillin-binding protein PBP 2A, respectively. Antibiotic sensitivity test was performed by the Kirby–Bauer disc diffusion method. The Clinical and Laboratory Standard Institute guidelines as CLSI M100-S27 was used for analysis of the data.ResultsThirty-five percent of our samples were detected contaminated with of the S. aureus strains. Subsequently, antibiotic resistance was observed in the S. aureus contaminated samples. Among our samples, the highest rates of resistance were determined against ampicillin (97.1%), oxacillin (88.6%), and penicillin (88.6%), respectively. Three genes including mecA, sea, and tsst from six virulence genes were detected in 17% of S. aureus strain-contaminated samples by multiplex PCR. The sed, etb and eta genes were detected in the 2.9%, 11.4% and 5.7% of our samples, respectively.ConclusionThe results show that S. aureus related contamination is high in the raw beef for retail sale and prevalent S. aureus strains are resistant to all antibiotics used. Also, our results have demonstrated that there is a high risk for food poisoning caused by antibiotic resistant S. aureus in the raw beef and it may establish public health issues. Genes encoding for both heat-resistant and nonresistant toxicity factors were detected in the antibiotic resistant S. aureus strains and shown the highly pathogenic. Finally, our study is ensuring to need proper hygienic conditions during beef’s preparation and sale.

Molecular detection of virulence genes in Staphylococcus aureus isolated from wild pigeons (Columba domestica livia) in KwaZulu-Natal in South Africa

The current study aimed to determine virulence determinants among S. aureus isolated from wild pigeons and houseflies around hospital areas in the Greater Durban area, South Africa. Following enrichment and bacterial growth, DNA extraction using the boiling method was performed. Overall, 57 out of 252 samples (22.6%) were positive for S. aureus. Six known virulence genes were tested, where five known virulence determinants were positive and none of the S. aureus isolates were positive to coagulase (coa) gene. The highest prevalence rates were found in the genes encoding haemolysins, with the hla and hld genes having 8 (14%) and 9 (15.8%) positive isolates respectively. The sea, LukS/F-PV, and spa genes had 5 (8.8%), 4 (7%), and 2 (3.5%) positive isolates respectively. These results demonstrated the detection of pathogenic S. aureus from hospital environment in Durban, South Africa which may account for the emergence staphylococcal infections. The findings of the present study highlights the significant role of wild pigeons and houseflies as potenital infectious disease vectors in a One Health context.

Whole-Genome Sequencing Analysis of Non-Typhoidal Salmonella Isolated from Breeder Poultry Farm Sources in China, 2020-2021.

Non-typhoidal salmonellosis is a dangerous foodborne disease that causes enormous economic loss and threatens public health worldwide. The consumption of food, especially poultry or poultry products, contaminated with non-typhoidal Salmonella (NTS) is the main cause of human salmonellosis. To date, no research has identified the molecular epidemiological characteristics of NTS strains isolated from breeder chicken farms in different provinces of China. In our study, we investigated the antimicrobial resistance, phylogenetic relationships, presence of antimicrobial resistance and virulence genes, and plasmids of NTS isolates recovered from breeder chicken farms in five provinces of China between 2020 and 2021 by using a whole-genome sequencing (WGS) approach and phenotypic methods. All sequenced isolates belonged to six serovars with seven sequence types. Nearly half of the isolates (44.87%) showed phenotypic resistance to at least three classes of antimicrobials. Salmonella enterica serotype Kentucky harbored more antimicrobial resistance genes than the others, which was highly consistent with phenotypic resistance. Furthermore, the carried rate of 104 out of 135 detected virulence genes was 100%. Overall, our WGS results highlight the need for the continuous monitoring of, and additional studies on, the antimicrobial resistance of NTS.

Detection of Virulence Genes, Antimicrobial Susceptibility and Pathogenicity of Aeromonas veronii Isolates from Labeo rohita and Catla catla

Background: Aeromonas veronii is the most common bacterium responsible for diseases in freshwater fish rearing systems. Multiple factors can be involved in the virulence processes of Aeromonas bacteria. Hence, the purpose of the present investigation was to evaluate the virulence genes, antimicrobial susceptibility and pathogenicity of A. veronii isolated from freshwater fishes. Methods: In this investigation, we isolated A. veronii from cultured freshwater fishes, Labeo rohita and Catla catla. A. veronii was identified by bacterial staining and culture characteristics. In addition, Polymerase chain reaction (PCR) was used to evaluate the distribution of nine virulence genes including aerolysin, cytotonic enterotoxin (442, 272 bp), elastase, enolase, flagellin, lipase, serine protease and DNase. Result: A total of 88 A. veronii strains were isolated, which includes 56 strains from rohu and 33 strains from catla. The strains were Gram-negative, rod-shaped, motile bacteria and the colonies are yellow on blood agar. All the A. veronii strains were positive for at least one or more virulence genes tested. The isolates carried more virulence genes, especially in the combination of aer,alt, ela, lip, AhyB genes were found to be more virulent. Antimicrobial susceptibility to 17 antibiotics was determined and the strains of A. veronii showed 100% resistance to tetracycline, pencillin and â-lactum group of antibiotics. In addition, multiple antibiotic resistance (MAR) indexes ranged from 0.24 to 0.76, suggesting that they originated from high risk contaminated zones were (W. Godavari and Nellore districts) antimicrobials are often used.

Detection of virulence genes and multidrug resistance in Pseudomonas aeruginosa clinical isolates from a public hospital in Lagos, Nigeria

Pseudomonas aeruginosa is a main cause of nosocomial infections and is classified as a priority critical pathogen of global concern. Multidrug resistance and virulence genes attributed to the pathogenicity of the organism has resulted in failed therapies, linked to increased morbidity and mortality in infected patients. The aim of this study was to determine the virulence genes and multidrug resistant P.aeruginosa in clinical specimens from a secondary health care center laboratory in Lagos, Nigeria. MethodologyA total of three hundred and sixty-seven clinical samples were examined with conventional methods and Microbact GNB12A. Forty- seven (47) P. aeruginosa strains were identified. Antimicrobial susceptibility test was performed by the standard disk diffusion method following CLSI guidelines. Identification of P. aeruginosa was done by amplification of oprL and oprI genes. Monoplex and multiplex PCR assays were used for detecting virulence genes (lasB, toxA, exoU and nan1). Descriptive statistical methods were used to analyze the distribution of P. aeruginosa by sample sources, MDR patterns by number of isolates and virulence gene carriage by MDR (≥3classes of antibiotics). ResultsA total of forty-seven (47) isolates were confirmed as P. aeruginosa. The isolates showed pan-resistance to cefuroxime and ceftazidime. An elevated level of resistance was also seen in amoxycillin-clavulanic acid, ceftriaxone, cefixime and meropenem. Susceptibility to Amikacin was 95.7 %. Multidrug resistance was seen in forty (85.1 %) of the isolates. P. aeruginosa (100 %) were confirmed with oprL and 74.5 % oprI genes. All isolates (100 %) had lasB, toxA and exoS virulence genes while nan1 was detected in 19.1 % isolates. ConclusionsDiverse and multiple virulence genes were distributed among the multidrug resistant isolates. Understanding the factors contributing to pathogenicity and antibiotic resistance will aid in the treatment of infection. It will also promote judicious use of antimicrobials.

Seasonal effect on the prevalence of virulence genes of non-O157 Verotoxic E.coli serogroups in faeces of cattle calves

Calves faecal samples (n=216) were investigated to observe the effect of seasons on the prevalence of serogroups and virulence genes of non-O157 VTEC. A total of 177 (81.94%) E. coli were isolated and 32 (14.81%) were identified as VTEC and serotyping resulted in 13 different non-O157 ‘O’ serogroups. The prevalence of serogroups and their virulence genes was found to be influenced by seasons and highest number were shed in summer (22.22%) followed by rainy (13.88%) and winter season (8.33%), respectively. A higher prevalence of O9 and O11 serogroups (25% each) was observed in summers. Molecular detection of virulence genes revealed the overall prevalence of vt1 to be 37.5%, vt2 43.8%, (vt1+vt2) 18.8%, eaeA 21.9% and hlyA 34.4% genes. Dominance of hlyA 50% was observed in summers, whereas vt1 and vt2 were more prevalent during rain (50% each). The study revealed the link between the occurrence of hlyA gene and O9, O11 serogroups in summers as both the serogroups were hlyA gene bearer. This association might be responsible for more VTEC outbreaks in summers. So, faecal contamination of raw milk seems to pose greater threat of non-O157 VTEC outbreak during hotter and humid months.

Detection of mecA positive staphylococcal species in a wastewater treatment plant in South Africa

We investigated the prevalence of antibiotic resistant staphylococci and detection of resistant, virulence, and Spa genes in a South African wastewater treatment plant. Species identified were Staphylococcus aureus, S. lentus, S. arlettae, S. cohnii, S. haemolyticus, S. nepalensis, S. sciuri (now Mammaliicoccus sciuri), and S. xylosus. Isolates showed high resistance to methicillin (91%), ampicillin (89%), ciprofloxacin (86%), amoxycillin (80%), ceftazidime (74%), and cloxacillin (71%). Multiple antibiotic resistance (MAR) index for the isolates exceeded 0.2 (0.50–0.70). Among the isolates, 77% were mecA-positive. All S. aureus strains were positive for nuc and 7 Spa gene types. The present study highlights possibility of treated wastewaters being potential reservoir for antibiotic-resistant staphylococci. This is a cause for concern as wastewater effluents are decanted into environmental waters and these are, in many cases, used for various purposes including recreation (full contact), religious (full body submersion), and drinking water for some rural communities and water for livestock.

Occurrence and molecular characterization of Salmonella Typhimurium and Salmonella Enteritidis isolates from contaminated food samples from Palestine

Salmonella is one of the most frequently isolated foodborne pathogens. It is of major public health concern worldwide. Poultry meat and eggs represent an important source of Salmonellae organism for consumer health. This study aimed to evaluate the occurrence of Salmonella enterica serotype Typhimurium and Enteritidis using multiplex PCR (mPCR) among isolates collected from the local market and to assess genetic relationships between isolates of S. Typhimurium, which was the only serotype isolated from the tested food samples. This was done using virulence factors profiling and fingerprint profiling by random amplified polymorphic DNA (RAPD-PCR) and repetitive sequence PCR (REP-PCR) using enterobacterial repetitive intergenic consensus (ERIC-PCR) and interspersed repetitive DNA sequence BOXAIR-PCR.
 The overall occurrence percentage of S. Typhimurium and S. Enteritidis out of 51 isolates was 54.9% and 0.0%, respectively. Only 13 out of 17 virulence genes were detected in these isolates. The occurrence of the detected virulence genes among these isolates was 100%, 50.0%,46.4%, 39.3%, 35.7%, 35.7%, 32.1%, 25.0%, 25.0%, 17.6%, 14.3%, 14.3%, 3.6% for invA, sopB, prgH, sitC, pefA, tolC, cdtB, msgA, sifA, iroN, spiA, ipfC and pagC, respectively. The remaining virulence genes were absent in all of the isolates. Based on the combination of the presence and absence of virulence genes, eight profiles were detected among these isolates, the most common genetic profile was V5 (each 32.1%). Based on this genetic profile at cut-off point 96.0%, both ERIC and BOX primers allowed for discrimination into 4 and 6 clusters or clones of 16 S. Typhimurium isolates, respectively. Results of PCR typing methods showed that, three strains clustered together using both ERIC-PCR and BOX-PCR typing methods and they had the same virulotype (V1), while other four strains also clustered together by both typing methods and had the same virulotype (V8).
 Contamination of food with Salmonellae especially with S. Typhimurium was high and indicated a bad microbiological quality of food. This emphasizes the need for rigorous public health and food safety methods to lower the human health hazard and risk associated with Salmonellae infection.

Shiga toxin-producing Escherichia coli (STEC) in meat and leafy greens available in the Swedish retail market – Occurrence and diversity of stx subtypes and serotypes

Shiga toxin-producing Escherichia coli (STEC) is a major cause of foodborne illness, ranging from mild diarrhea to permanent kidney failure. This study summarizes the results of four surveys performed at different time periods, which investigated the occurrence and characteristics of STEC in beef, lamb and leafy greens available in the Swedish retail market. Such data is required when assessing the public health risk of varying types of STEC in different foods, and for establishing risk management measures. Samples from domestic and imported products were collected based on their availability in the retail market. The occurrence of STEC was investigated in 477 samples of beef, 330 samples of lamb and 630 samples of leafy greens. The detection of virulence genes (stx1, stx2, eae) was performed using real-time PCR followed by the isolation of bacteria from stx-positive enriched samples using immunomagnetic separation or an immunoblotting method. All STEC isolated from the food samples was further characterised in terms of stx subtyping and serotyping through whole genome sequencing. STEC was isolated from 2 to 14 % of beef samples and 20 to 61 % of lamb samples, depending on the region of origin. STEC was not isolated from samples of leafy greens, although stx genes were detected in 11 (2 %) of the samples tested. In total, 5 of the 151 sequenced STEC isolates from meat contained stx2 and eae, of which 4 such combinations had the stx2a subtype. The stx2 gene, stx2a in particular, is strongly associated with serious disease in humans, especially in combination with the eae gene. The isolates belonged to 20 different serotypes. Two isolates from beef and one from lamb belonged to the serotype O157:H7 and contained genes for stx2 and eae. Overall, several combinations of stx subtypes were found in isolates from beef, whereas stx1c, either alone or together with stx2b, was the dominant combination found in STEC from lamb. In conclusion, STEC was rare in whole meat samples of domestic beef in the Swedish retail market, whereas such bacteria were frequently found in minced meat and whole meat samples of imported beef and domestic and imported lamb. Although the number of isolates containing genes linked to an increased risk of severe disease was low, beef and lamb in the Swedish retail market is a common source of human exposure to potentially pathogenic STEC.

High Incidence of Metastatic Infections in Panton-Valentine Leucocidin-Negative, Community-Acquired Methicillin-Resistant Staphylococcus aureus Bacteremia: An 11-Year Retrospective Study in Japan.

Panton-Valentine leucocidin (PVL)-negative community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was originally disseminated in Japan and has since replaced healthcare-associated MRSA (HA-MRSA). However, the clinical characteristics of CA-MRSA bacteremia (CA-MRSAB) compared with those of HA-MRSA bacteremia (HA-MRSAB) are unknown. We aim to clarify differences and investigate associations between the clinical manifestations and virulence genes associated with plasma-biofilm formation in PVL-negative CA-MRSA. From 2011 to 2021, when CA-MRSA dramatically replaced HA-MRSA, 79 MRSA strains were collected from blood cultures and analyzed via SCCmec typing and targeted virulence gene (lukSF-PV, cna, and fnbB) detection. The incidence of metastatic infection was significantly higher in CA-MRSAB than in HA-MRSAB. PVL genes were all negative, although cna and fnbB were positive in 55.6% (20/36) and 50% (18/36) of CA-MRSA strains and 3.7% (1/27) and 7.4% (2/27) of HA-MRSA strains, respectively. cna and fnbB carriage were not associated with the development of metastatic infections in MRSAB; however, the bacteremia duration was significantly longer in CA-MRSAB harboring cna. CA-MRSAB may be more likely to cause metastatic infections than HA-MRSAB. Since CA-MRSA is dominant in Japan, suspected metastatic infection foci should be identified by computed tomography, magnetic resonance imaging, and echocardiography when treating MRSAB.

Molecular detection of virulence genes in pseudomonas aeruginosa isolated from different clinical infections

Objective: To isolate pseudomonas aeruginosa from different clinical specimens, and to detect virulence factors.Method: The cross-sectional study was conducted at Al-Zharr Hospital and private clinics in Kut city of Wasit provincein Iraq from October 15, 2021, to April 30, 2022, and comprised samples obtained from different patient sites, such aswound swabs, ear swabs, urine samples, burn swabs and respiratory tract swabs. The samples were subjected tomolecular testing using, among other tools, polymerase chain reaction. The isolates were cultured on different media,such as blood agar, MacConkey agar and cetrimide agar, and were further diagnosed by biochemical testing. Datawas analysed using SPSS 20.Results:Of the 212 samples, there were 70(33%) wound swabs, 53(25%) ear swabs, 41(19.3%) urine samples, 33(15.6%)burn swabs and 15(7%) respiratory tract swabs. Pseudomonas aeruginosa was identified in 54(25.47%) isolates viaculture-based characterisation. All 54(100%) isolates were positive for mexR and toxA on polymerase chain reaction,44(81.48%) were positive for exoU gene, 43(79.62%) for exoT gene, 36 (66.66) for exoT gen, 35(64.81%) for plcH, geneand 14(25.92 %) for exoS gene.Conclusions: Wound swabs were found to be the most common site for pseudomonas aeruginosa, followed by earswabs and urine samples.Keywords: Pseudomonas aeruginosa, Virulence, Agar, Cetrimonium, Virulence, Burns, Polymerase chain reaction, DNA.

Molecular epidemiology and antibiotic resistance of Pseudomonas aeruginosa isolated from blood in a hospital in Shandong Province from 2014 to 2021

Objective: To identify the antibiotic resistance, virulence genes, and sequence types of Pseudomonas aeruginosa (P. aeruginosa) strains isolated from blood. Methods: From November 2014 to December 2021, a total of 94 nonrepetitive P. aeruginosa isolates were obtained from blood samples of patients at the First Affiliated Hospital of Shandong First Medical University in Shandong Province, China. The bacteria were identified using matrix-assisted laser desorption ionization time of flight mass spectrometry. Antibiotic resistance of the P. aeruginosa isolates was detected using Vitek 2 Compact system. Polymerase chain reaction (PCR) was conducted for the 18 virulence genes, and multi locus sequence typing (MLST) was performed to identify the sequence types of the P. aeruginosa strains. The resistance rates and distributions of virulence genes between carbapenem resistant pseudomonas aeruginosa (CRPA) and carbapenem susceptible pseudomonas aeruginosa (CSPA) isolates were compared using the Chi-square test. Results: Among 94 P. aeruginosa isolates, 19 (20.2%) isolates were found to be multidrug resistant (MDR) bacteria, of which 17 were CRPA isolates and 2 were CSPA isolates. All strains contained more than 10 virulence genes. Except for exoU gene, the detection rate of other genes was above 83%. MLST analysis revealed a total of 66 different STs, including 59 existing STs and 7 novel STs. Among them, ST244 (n=11, 11.7%) and ST270 (n=7, 7.4%) were the dominant STs. Although these two types of isolates harbored the same virulence genes, the resistance rates to carbapenem were different. 54.5% (6/11) ST244 isolates were CRPA but all 7 ST270 isolates were CSPA. Conclusion: Although the resistance rates of P. aeruginosa strains isolated from blood were at a low level, some MDR and CRPA isolates were detected. As the high virulence gene detection rates and genetic diversity were found for P. aeruginosa strains isolated from blood, close attention should be paid to avoid transmission and outbreaks.