Prevalence of USP and hlyA Genes and Association with Drug Resistance in Uropathogenic Escherichia coli Isolated from Patients in a Tertiary Hospital from Southeast China.

E. coli was cultured from the urine of patients from the tertiary hospital located in Southeast China from 2017 to 2019. The species were identified, drug sensitivity test was performed, and the presence of the virulence genes USP and hlyA was determined. A total of 483 strains of E. coli were isolated, including 132 from patients with urinary tract infection (UTI). The resistance to ciprofloxacin was more common in non-UTI patients, while resistance to gentamycin was significantly higher in the UTI group. In the UTI group, the proportions of isolated bacteria with the virulence USP (40.15%) and hlyA (8.33%) genes were significantly higher than in the non-UTI group (19.60 and 2.56%, respectively). The rate of resistance of E. coli toward levofloxacin in the USP+ group was significantly (p<0.05) higher than in the USP- group. Thus, we revealed the differences in the rate of drug resistance and prevalence of USP and hlyA between the UTI and non-UTI groups. Furthermore, the presence of the USP gene was found to be associated with greater resistance to levofloxacin.

Establishment and application of a rapid visual detection method for Listeria monocytogenes based on polymerase spiral reaction (PSR).

Listeria monocytogenes is a common foodborne pathogen that presents in various food products, posing important threat to public health. The aim of this study was to establish a rapid and sensitive method with visualization to detect L. monocytogenes based on polymerase spiral reaction (PSR). Primers targeting conserved hlyA gene sequence of L. monocytogenes were designed based on bioinformatics analyses on the current available L. monocytogenes genomes. The isothermal amplification PSR can be completed under constant temperature (65ᵒC) within 60 min with high specificity and sensitivity. Twenty-five reference strains were used to evaluate the specificity of the developed reaction. The results showed that the sensitive of the reaction for L. monocytogenes in purified genomic DNA and artificially contaminated food samples were 41 pg/µL and 103 CFU/mL, respectively. It was 100-fold more sensitive than conventional PCR. In conclusion, this novel PSR method is rapid, cost-efficient, timesaving, and applicable on artificially contaminated food samples, providing broad prospects into the detection of foodborne microbes with the promising on-site inspection.

First detection of Shiga toxin producing Escherichia coli O157:H7 (HlyA gene) and enumeration of most probable number (MPN) of fecal coliforms and Escherichia coli in cage cultured oysters (Crassostrea iredalei) and water from southern Malaysia

Aims@#Escherichia coli O157:H7 is known to be transmitted via fecal-oral route, where water plays a role in the transmission process. Oysters as bivalves, bio accumulate pathogens from the water through filter feeding and are suspected to play a role as disease transmission vector. In Malaysia, the data on oyster’s microbiological quality are limited. Hence, it was vital to conduct oyster related studies in Malaysia. The main objectives of this study include the enumeration of most probable number (MPN) of fecal coliforms and E. coli and isolation of E. coli from oyster (Crassostrea iredalei) and water sample for the detection of 16S rRNA and HlyA (Hemolysin A) genes of E. coli O157:H7. @*Methodology and results@#A total of 120 oysters and water samples (n=6) were collected from a fisherman village located in southern Malaysia. Total fecal coliforms and E. coli were determined using the MPN procedure. Colonies of E. coli were identified based on Gram staining, biochemical test, and PCR detection for the presence of 16S rRNA and HlyA gene of E. coli O157:H7. The enumeration results showed that the MPN of the fecal coliforms and E. coli found in the collected oyster samples do not meet the standard to be directed for human consumption (0.72 ± 0.19 × 104 MPN/100 g and 0.13 ± 0.03 × 10 4 MPN/100 g, respectively). The PCR assays showed that 16 out of the 104 (15.38%) of E. coli isolated from water and oysters showed the presence of HlyA gene. The phylogenetic tree analysis showed there were genetic relationships between the HlyA gene of the E. coli isolated in this study with the ones isolated from calf and human faeces.@*Conclusion, significance and impact of study@#The detection of Shiga toxin producing E. coli O157:H7 (HlyA gene) in cage cultured oysters (C. iredalei) and water from southern Malaysia was first time reported here. In the future, more study can be conducted to study the expression of the HlyA gene and confirm of its identity as E. coli O157:H7 using different target genes such as eaeA (encodes a 94 kD outer membrane protein called intimin) and Stx1 (Shiga toxin, Shigella dysenteriae type 1).

The Novel Multiple Inner Primers-Loop-Mediated Isothermal Amplification (MIP-LAMP) for Rapid Detection and Differentiation of Listeria monocytogenes.

Here, a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MIP-LAMP), was devised and successfully applied to detect Listeria monocytogenes. A set of 10 specific MIP-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene. The MIP-LAMP assay efficiently amplified the target element within 35 min at 63 °C and was evaluated for sensitivity and specificity. The templates were specially amplified in the presence of the genomic DNA from L. monocytogenes. The limit of detection (LoD) of MIP-LAMP assay was 62.5 fg/reaction using purified L. monocytogenes DNA. The LoD for DNA isolated from serial dilutions of L. monocytogenes cells in buffer and in milk corresponded to 2.4 CFU and 24 CFU, respectively. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by adding Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel electrophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MIP-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MIP-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.

Label-free impedimetric detection of Listeria monocytogenes based on poly-5-carboxy indole modified ssDNA probe.

Listeria monocytogenes is a life threatening pathogenic bacteria concerned with human health. The accurate and rapid detection of L. monocytogenes is required for preventing of listeriosis. In this study, DNA sensing probe based on conducting polymer poly-5-carboxy indole (5C Pin) was developed for the detection of L. monocytogenes hlyA gene responsible for pathogenicity. The probe sequences (24 mer ssDNA) were covalently immobilized on 5C Pin via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS). The probe having ssDNA was further hybridized with the target DNA sequence. Electrochemical impedance spectroscopic study was carried out to determine the extent of DNA hybridization over the probe. Significant change was observed in the impedance spectra before and after hybridization of ssDNA immobilized over the probe with the target DNA. RCT (charge transfer resistance) was estimated from the Nyquist plot (impedance plot) for target DNA (hlyA gene) in the solution. RCT vs. logarithmic concentrations of the target (genomic) DNA plot showed a linear range (1 × 10(-4) to 1 × 10(-12)M) in case hybridization was performed under optimized conditions. The method proposed, is simple, free from any label, and highly sensitive for the detection of L. monocytogenes in environmental and clinical samples.

'Prevalence and antimicrobial susceptibility of Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains from raw meat and meat products in Zaria, Nigeria.

UNLABELLED: The bacterial genera Listeria and Staphylococcus have been frequently isolated from food products and are responsible for a number of animal and human diseases. The aim of the study was to simultaneously isolate and characterize L. monocytogenes and Staphylococcus species from 300 samples of raw meat and meat products, to determine the susceptibility of the organisms to commonly used antimicrobial agents and to determine the presence of haemolysin A (hyl) virulence gene in L. monocytogenes and staphylococcal cassette chromosome mecA (SCCmec) gene in the Staph. aureus isolates using PCR. Of the 85 Listeria isolates tested, 12 L. monocytogenes were identified and tested for their sensitivity to 14 antimicrobial agents. All the 12 isolates (100%) were resistant to nine antimicrobial agents, but however sensitive to gentamicin. Only one isolate was found to harbour the hylA gene. Twenty-nine isolates were confirmed as Staph. aureus by the Microbact 12S identification system and were all presumptively identified as methicillin-resistant Staph. aureus species using oxacillin-resistant Staph. aureus basal medium (ORSAB). The 29 Staph. aureus isolates were tested for their sensitivity to 16 antimicrobial agents, and 11 were resistant to methicillin. None of the 11 Staph. aureus isolates harboured the methicillin resistance, mecA gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes and Staphylococcus aureus are important agents of foodborne diseases. Occurrence of these infectious agents was established in meat and meat products in Zaria, Nigeria. Majority of isolates obtained from this study, displayed multidrug resistance to commonly used antimicrobial agents, including methicillin resistance among the Staph. aureus isolates. The potential virulence of L. monocytogenes found in ready-to-eat food was documented by the carriage of hly A gene by one of the isolates. A different mechanism of methicillin resistance or different homologue of mec A gene may be circulating among Nigerian isolates.