hlyA Gene Research Articles

Networking and co-occurrence of virulent and multidrug resistant environmental bacteria in different aquatic systems: A gap in MDR-virulence transfer?

Co-occurrence of resistance and virulence is often overlooked in aquatic bacteria as environmental reservoirs, while transmission of these characteristics to clinically significant strains present unforeseen problems in future. In this investigation, environmental bacteria identified concurrently from multiple aquatic habitats viz., groundwater, canal, river and coastal waters were profiled for antibiotic resistance, metal tolerance, virulence factors and genes coding for these determinants. Strains from polluted river and canal exhibited higher resistance and virulence, especially Pseudomonas gessardii and P. fluorescens displayed high antibiotic resistance index (ARI > 0.6-0.8) with Alkaline Protease and Phospholipase production. Opportunistic pathogens including Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, Corynebacterium and Comamonas testosteroni expressed all three virulence factors with relatively low resistance. However, V. vulnificus and V. alginolyticus exhibited multiclass antibiotic resistance (5/6 classes). Metagenomic analysis revealed that genes corresponding to beta-lactam resistance were significantly higher (p < 0.05) in freshwater than seawater, while multidrug resistance gene were higher (p < 0.05) in seawater. In all aquatic bodies, abundant virulence genes belonged to secretion system proteins followed by motility related genes. Culturable bacteria revealed differential distribution of positive and negative correlation between 31 targeted genes with expressed resistance and virulence. Among Acinetobacter, significant positive correlation was found between Phospholipase production, other virulence genes (OVGs) and resistance to DNA Synthesis Inhibitors (DSI). In Pseudomonas, positive correlation was detected between toxin genes (toxA, eta, hlyA and stx) and resistance to cell wall synthesis inhibitors (CSI) as well as with OVGs and adhesion genes (eae, afa, papC and papA). Network analysis displayed unique clustering of genes ncc, arsB, strA, merA and intI dominated by non-pathogens and distinct clustering of genes pho, erm, nfsA, trh, lasB, tdh and invA by Vibrio. This investigation extends insight on co-occurring resistance and virulence in aquatic reservoir bacteria that could pose serious threats to public health in future.

Multidrug resistant enterohaemorrhagic Escherichia coli serogroups in the faeces of hunted Wildlife, Abeokuta, Nigeria.

Wildlife play significant roles in the dissemination and zoonotic transmission of pathogens. The enterohaemorrhagic Escherichia coli (EHEC) are associated with complicated cases of food-borne illnesses. This study investigated the presence of EHEC serogroups (O26, O45, O103, O145, O91, O111, O128, O121 and O157) in wildlife species: cane rats (Thryonomys swinderianus), royal antelope (Neotragus pygmaeus), African giant rats (Cricetomys gambianus) and waterbuck (Kobus ellipsiprymnus). EHEC and non-EHEC isolates from these wildlife sources were tested for susceptibility to antimicrobial agents. Overall, 127 (83.0 %) of 153 samples yielded E. coli. Nine (5.9%) samples were positive for EHEC belonging to three serogroups as follows; O26 (n=2), O111 (n=2) and O103 (n=5). The EHEC isolates were from cane rats (n=6) and royal antelope (n=3) and possessed virulence-associated genes stx1 (77.8%), stx2 (100.0%), eaeA (100.0%) and hlyA (100.0%). Overall, 127 E. coli isolates showed resistance to ampicillin (99.2%), ceftiofur (90.6%), tetracycline (90.0%), cephalexin (87.4%), cefotaxime (50.4%), streptomycin 42.5%, ceftazidime (41.7%), nalidixic acid (37.0%), ciprofloxacin (43.6%), amoxicillin/clavulanic acid (32.3%), gentamicin (27.6%), sulphamethoxazole/trimethoprim (25.2%), norfloxacin (17.3%) and chloramphenicol (11.0%). The roles of wildlife in the dissemination and transmission of antimicrobial resistant and zoonotic bacteria should not be neglected for effective preventive and control strategies.

LISTERIA MONOCYTOGENES IN READY-TO-EAT CHICKEN PRODUCTS, THEIR ANTIBIOTIC RESISTANCE AND VIRULENCE GENES

Ready-to-eat (RTE) meat products are food items that are extremely popular worldwide. Albeit documented with the alarming prevalence of pathogenic multidrug-resistant Listeria species in these food items worldwide, studies from the perspective of Bangladesh are absent. This study was devised with the aim to detect pathogenic Listeria monocytogenes in some of the most popular RTE meat products in Dhaka, Bangladesh. Thirty-nine such samples were investigated using biochemical tests and the Polymerase Chain Reaction (PCR) analysis. Following confirmatory tests and detection of Listeria species, the isolates were subjected to the Kirby-Bauer disc diffusion test to investigate their antibiotic-susceptibility patterns against some of the most commonly used antibiotics to treat listeriosis – the infection caused by Listeria. Five Listeria species were detected using biochemical and PCR tests. This constituted an overall prevalence rate of 12.8% (n = 39). Four out of the five Listeria species were concluded to be L. monocytogenes, while the remaining one was an L. innocua isolate. PCR analyses revealed all four of the L. monocytogenes isolates to have the virulence genes hlyA, plcB and actA, although no polymorphism was observed for these genes. Despite the presence of these pathogenic genes, antibiotic susceptibility tests showed promising results as some of the most commonly prescribed drugs against listeriosis were highly effective against the isolates. Even though the isolates showed little to no antibiotic resistance against the antibiotics used in the study, the presence of three virulence genes in all four L. monocytogenes isolates is still a cause for concern. Even more so, the prevalence of these pathogenic strains in a food item that is increasing in popularity in a country like Bangladesh, where awareness and surveillance against listeriosis is limited, can have dire consequences should an outbreak occurs. Bioresearch Commu. 9(1): 1160-1169, 2023 (January)

Some Virulence Genes Profile of Escherichia coli Stains Isolated from Urinary Tract Infection in Iraqi patients

Background: The urinary tract infection (UTI) is the most frequent bacterial infectious illness seen in clinical practice, accounting for considerable morbidity and high medical expenses. Escherichia coli is the most prevalent bacterium causing UTIs. The expression of a broad range of virulence factors by E. coli contributes to the severity of UTI. This research investigated the function of E. coli virulence determinants in the pathogenesis of UTI. The aim of the present study is to identify virulence genes in Uropathogenic E. coli which are responsible for UTI by isolation and identification of major microorganisms (Uropathogenic E. coli) that cause UTI and detection of antimicrobial susceptibility of the bacterial isolates. The determination of some virulence uropathagant genes such as cnf, hly, and fimH genes by PCR. Material and Methods: From March 2022 to June 2022, approximately 200 clinical urine samples were collected from patients suffering from UTI of both genders; (160) urine samples from females and (40) urine samples from males in age groups ranging from 15–50 years who attended Al-Sadr Teaching Hospital, Al-Zahrawi Surgical Hospital, and Misan Hospital for child and childbirth in Misan city, Iraq. Results: In the current study, E.coli has some virulence factors such as cnf, hly, and fimH for (40) E.coli stains were reported as a positive 4(10%), 30(75%), and and 13(32.5%) respectively. Conclusions: Our findings suggest that looking into the bacterial pathogenicity linked to UTIs might help doctors provide better care.

Development and study of the performance of a reagent kit for the identification of &lt;i&gt;Vibrio cholerae&lt;/i&gt; and &lt;i&gt;Vibrio parahaemolyticus&lt;/i&gt; by multilocus allele-specific polymerase chain reaction in clinical trials

BACKGROUND: Cholera belongs to the group of infections, and the fight against it is regulated by the WHO International Health Regulations (2005). To date, several test systems have been developed for the polymerase chain reaction (PCR) detection of Vibrio cholerae strains. However, no PCR test systems have been registered for detecting V. parahaemolyticus DNA in clinical material in Russia.&#x0D; AIM: To conduct clinical trials and evaluate the performance of the Reagent kit for identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR (kit of reagents Vibrio-screen FL) for the state registration of the specified medical device.&#x0D; MATERIALS AND METHODS: The study presents the results of the clinical trials for the reagent kit Vibrio-screen FL that has been developed to optimize the means and methods of identification and differentiation of pathogens of dangerous infections based on data on the diversity of genomes. Clinical trials were conducted at the Head Center of Hygiene and Epidemiology of the FMBA of Russia (license from 02.12.2013 No. ФС-99-01-008342; permission of the Ministry of Health from 16.05.2013 No. 300n) in accordance with the program of clinical trials No. 20.ПМКИ-042/10.20 from 23.09.2020 (Clinical Trial Certificate No. 01.AK-042/10.20 dated 12.10.2020). The clinical trials of the developed reagent kit Vibrio-screen FL used clinical strains of microorganisms of the genus Vibrio and other microorganism species as target analytes. Bacteriological method and multilocus PCR with hybridization-fluorescence detection were used.&#x0D; RESULTS: The reagent kit Vibrio-screen FL is a medical device for professional use in clinical laboratory diagnostics. The analytical sensitivity of the reagent kit Vibrio-screen FL was at least 106 copies of the SecY gene when determining belonging to the genus Vibrio; to the species V. cholerae, at least 106 copies of the hlyA gene; and to the species V. parahaemolyticus, at least 106 copies of the vppC gene. When determining the analytical specificity, no non-specific reactions with DNA samples of other microorganism species were found. A medical device for in vitro diagnostics A reagent kit for the identification of V. cholerae and V. parahaemolyticus by multilocus allele-specific PCR (Vibrio-screen FL) meets the requirements of the intended use, which allows it to be used for the differentiation of V. cholerae and V. parahaemolyticus strains isolated from a cultured clinical samples by multilocus allele-specific polymerase chain reaction with hybridization-fluorescent detection. The registration certificate No. РЗН 2021/13360 from 08.02.2021 was received. By the order of Roszdravnadzordated 08.02.2021 No. 1041, a reagent kit Vibrio-screen FL by the Rostov-on-Don Anti-Plague Institute of Rospotrebnadzor was admitted to distribution on the territory of the Russian Federation.&#x0D; CONCLUSIONS: The reagent kit Vibrio-screen FL is a high-quality, effective, and safe diagnostic medical device for in vitro diagnostics with sufficient diagnostic sensitivity and specificity to identify specific fragments of Vibrio spp., V. cholerae, and V. parahaemolyticus nucleic acids. These allows it to be used in clinical laboratory diagnostics for epidemiological analysis and registration of diseases caused by pathogenic vibrions.

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

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

Distribution and expression of virulence genes (hlyA, sat) and genotyping of Escherichia coli O25b/ST131 by multi-locus variable number tandem repeat analysis in Tehran, Iran.

Escherichia coli ST131 is a pandemic clone with high antibiotic resistance, and it is a major causative agent of urinary tract infection (UTI) and bloodstream infections. This study evaluated the distribution and expression of virulence genes and genotyping of E. coli O25b/ST131 by Multi-locus variable number tandem repeat analysis (MLVA) method among UTI in patients at Tehran hospitals, Iran.A total of 107 E. coli isolates were collected from UTI patients. Polymerase chain reaction (PCR) amplification of the pabB gene was used to identify E. coli O25b/ST131 and the prevalence of sat and hlyA virulence genes was also analyzed. The microtiter method quantified biofilm formation ability in E. coli O25b/ST131. The Real-Time PCR (qRT-PCR) was performed to evaluate the expression of sat and hlyA genes. Finally, MLVA was performed for E. coli O25b/ST131 genotyping by targeting seven tandem repeats. SPSS-16 software was used for statistical analysis. Molecular study showed that 71% of isolates carried the pabB gene and were considered E. coli O25b/ST131 strains. Also, 45.8% and 17.8% of isolates carried sat and hlyA genes, respectively. The 57.9% isolates had biofilm formation ability. Expression of the studied virulence genes showed an increase in strong biofilm producing E.coli O25b/ST131 strains. A total of 76 (100%) E. coli O25b/ST131 strains were typed by the MLVA method.High prevalence of E. coli O25b/ST131 isolates in UTI patients can be a serious warning to the treatment due to the high antibiotic resistance rate, expression of virulence genes, and biofilm formation.

Synthetic Analogs of Antimicrobial Peptides from Limulus Inhibit the Growth of Listeria monocytogenes by Increasing Cell Membrane Permeability and Suppressing Virulence Genes

Listeria monocytogenes is a foodborne pathogen that can cause listeriosis in humans and animals. It is of significant concern to the food industry as it can grow at low temperatures. Aggravatingly, only some of the commonly used food preservatives can effectively inhibit the growth of LM. In this study, the effectiveness of synthetic analogs of antimicrobial peptides from Limulus in inhibiting the growth of LM was studied. As determined by the Kirby–Bauer disc diffusion method, the diameters of the inhibition zones produced by the synthetic antimicrobial peptides ranged from 8.24 mm to 8.86 mm, and the minimum inhibitory concentrations (MICs) of the peptides ranged from 40 μg/mL to 160 μg/mL. At their MICs, initially, the synthetic antimicrobial peptides exhibited bacteriostatic effects on LM. They permeabilized the cell membrane of the bacterium and suppressed virulence genes (the inlA, prfA, and hly genes) in the bacterium. However, the bacteriostatic effects were effective only for 1 h, after which the bacterium slowly became resistant to them. After 6 h, the bacterium resumed its growth. Although cells in treatment groups resumed their growth after 6 h, the growth of the cells was inhibited compared to the growth of cells in the control group. Further studies are necessary to reduce the resistance of LM to the antibacterial effects of the synthetic antimicrobial peptides.

Characterization of virulence factors and antibiotic resistance pattern of uropathogenic Escherichia coli strains in a tertiary care center.

Background: Urinary tract infection(UTI) is one of the commonly prevalent bacterial infection in humans.The uropathogenic E. coli (UPEC) expresses a range of virulence factors that contribute to their pathogenicity . The emergence of multidrug resistance (MDR)-associated UTI is increasing.This study monitors the distribution of virulence factors among UPEC strains to note the antibiogram, outcome and type of associated UTI. Methods:A prospective cross-sectional time-bound study of six months was done on clinically significant urinary isolates of Escherichia coli. Detection of haemolysin production and serum resistance was done by phenotypic methods. Genotypic characterization of the virulence genes ( papC, iutA, hlyA, cnf1) was done by multiplex PCR. Demographic data, clinical history, antibiogram and type of UTI was collected from clinical case records. Results:75 E.coli isolates from patients with suspected UTIs were included. Females had a higher preponderance of UTI (66.7%). 93% of patients were adults and the remaining 7% were from paediatrics. 24 (32%) isolates showed haemolysis by plate haemolysis and all isolates were serum-resistant. Out of 75 isolates, 65 were positive for at least one of four targeted genes, while remaining ten isolates were negative for all four genes.Multidrug resistance was found in 40 (53.3%) isolates. 97.4% of the UTI cases had a favourable clinical outcome at discharge. Mortality due to urosepsis was 2.6%. Conclusion:Association of hemolysin production with resistance to imipenem and norfloxacin in UPEC strains was significant.Presence of hlyA gene is positively associated with ceftazidime resistance. Nitrofurantoin, piperacillin, tazobactam, and cefaperazone sulbactam are possible candidates for empirical therapy of UTIs. Drugs like aminoglycosides, carbapenems and fosfomycin may be used as reserve drugs in the treatment of MDR-UTI.However, inappropriate usage can increase antibiotic resistance. Hence proper selection of antibiotics in hospitals taking into account the local antibiogram is needed to reduce the emergence of antibiotic resistance.

Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

Exploring the Interactive Effects of Thymol and Thymoquinone: Moving towards an Enhanced Performance, Gross Margin, Immunity and Aeromonas sobria Resistance of Nile Tilapia (Oreochromis niloticus).

Plant-derived bioactive compounds with promising nutritional and therapeutic attributes (phytogenics) are among the top priorities in the aquaculture sector. Therefore, the impact of thymol (Thy) and/or thymoquinone (ThQ) on the growth, immune response antioxidant capacity, and Aeromonas sobria (A. sobria) resistance of Nile tilapia was investigated. Four fish groups were fed a control diet and three basal diets supplemented with 200 mg/kg diet of Thy or ThQ and a blend of both Thy and ThQ at a level of 200 mg/kg diet each. At the end of the feeding trial (12 weeks), the tilapias were challenged intraperitoneally with virulent A. sobria (2.5 × 108 CFU/mL) harboring aerolysin (aero) and hemolysin (hly) genes. The results revealed that tilapias fed diets fortified with a combination of Thy and ThQ displayed significantly enhanced growth rate and feed conversion ratio. Notably, the expression of the genes encoding digestive enzymes (pepsinogen, chymotrypsinogen, α-amylase and lipase) and muscle and intestinal antioxidant enzymes (glutathione peroxidase, catalase and superoxide dismutase) was significantly upregulated in Thy/ThQ-fed fish. An excessive inflammatory response was subsided more prominently in the group administrated Thy/ThQ as supported by the downregulation of il-β, il-6 and il-8 genes and in contrast, the upregulation of the anti-inflammatory il-10 gene. Remarkably, dietary inclusion of Thy/ThQ augmented the expression of autophagy-related genes, whilst it downregulated that of mtor gene improving the autophagy process. Furthermore, Thy/ThQ protective effect against A. sobria was evidenced via downregulating the expression of its aero and hly virulence genes with higher fish survival rates. Overall, the current study encouraged the inclusion of Thy/ThQ in fish diets to boost their growth rates, promote digestive and antioxidant genes expression, improve their immune responses and provide defense against A. sorbia infections with great economic benefits.

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.

Occurrence of Listeria spp. in Soft Cheese and Ice Cream: Effect of Probiotic Bifidobacterium spp. on Survival of Listeria monocytogenes in Soft Cheese.

Listeria monocytogenes is one of the most important emerging foodborne pathogens. The objectives of this work were to investigate the incidence of Listeria spp. and L. monocytogenes in soft cheese and ice cream in Assiut city, Egypt, and to examine the effect of some probiotic Bifidobacterium spp. (Bifidobacterium breve, Bifidobacterium animalis, or a mixture of the two) on the viability of L. monocytogenes in soft cheese. The existence of Listeria spp. and L. monocytogenes was examined in 30 samples of soft cheese and 30 samples of ice cream. Bacteriological analyses and molecular identification (using 16S rRNA gene and hlyA gene for Listeria spp. and L. monocytogenes, respectively) were performed on those samples. Additionally, Bifidobacterium spp. were incorporated in the making of soft cheese to study their inhibitory impacts on L. monocytogenes. Out of 60 samples of soft cheese and ice cream, 25 samples showed Listeria spp., while L. monocytogenes was found in only 2 soft cheese samples. Approximately 37% of soft cheese samples (11 out of 30) had Listeria spp. with about 18.0% (2 out of 11) exhibiting L. monocytogenes. In ice cream samples, Listeria spp. was presented by 47% (14 out of 30), while L. monocytogenes was not exhibited. Moreover, the addition of B. animalis to soft cheese in a concentration of 5% or combined with B. breve with a concentration of 2.5% for each resulted in decreasing L. monocytogenes efficiently during the ripening of soft cheese for 28 d. Listeria spp. is widely found in milk products. Probiotic bacteria, such as Bifidobacterium spp., can be utilized as a natural antimicrobial to preserve food and dairy products.

Characterization of virulence factors and antibiotic resistance pattern of uropathogenic Escherichia coli strains in a tertiary care center.

Background: Urinary tract infections (UTI) are the most prevalent bacterial infection in humans. The uropathogenic E. coli (UPEC) expresses a range of virulence factors that contribute to their pathogenicity . The emergence of multidrug resistance (MDR)-associated UTI is increasing. This study monitors the distribution of virulence factors among UPEC strains to note the antibiogram, outcome and type of associated UTI. Methods: A prospective cross-sectional time-bound study of six months was done on clinically significant urinary isolates of Escherichia coli. Detection of haemolysin production and serum resistance was done by phenotypic methods. Genotypic characterization of the virulence genes ( papC, iutA, hlyA, cnf1) was done by multiplex PCR. Demographic data, clinical history, antibiogram and type of UTI was collected from clinical case records. Results:75 E.coli isolates from patients with suspected UTIs were included. Females had a higher preponderance of UTI (66.7%). 93% of patients were adults and the remaining 7% were from paediatrics. 24 (32%) isolates showed haemolysis by plate haemolysis and all isolates were serum-resistant. Out of 75 isolates, 65 were positive for at least one of four targeted genes, while remaining ten isolates were negative for all four genes. Multidrug resistance was found in 40 (53.3%) isolates. 97.4% of the UTI cases had a favourable clinical outcome at discharge. Mortality due to urosepsis was 2.6%. Conclusion: Association of hemolysin production with resistance to imipenem and norfloxacin in UPEC strains was significant. Presence of hlyA gene is positively associated with ceftazidime resistance. Nitrofurantoin, piperacillin, tazobactam, and cefaperazone sulbactam are possible candidates for empirical therapy of UTIs. Drugs like aminoglycosides, carbapenems and fosfomycin may be used as reserve drugs in the treatment of MDR-UTI. However, inappropriate usage can increase antibiotic resistance. Hence proper selection of antibiotics in hospitals taking into account the local antibiogram is needed to reduce the emergence of antibiotic resistance.

Distribution Of Virulence Factors Genes And Their Association With Antibiotics Resistance In Streptococcus Agalactiae Isolated From Pregnant Women In Al Anbar City

Group B streptococci (GBS) are globally recognized to cause adverse pregnancy outcomes, such as stillbirths and miscarriages, andare one of the main causes of newborn sepsis and meningitis. The high resistance of GBS to antibiotics becomes difficult orimpossible to treat, becoming increasingly common, causing a global health crisis. It complicates their eradication, potentiallyleading to the development of chronic infections. A total of 181 specimens were obtained from pregnant women. Out of thesespecimens, 22 isolates were bacteriologically identified as S.agalactiae. They were collected from Al-Anbar Province hospitals.Twenty-two isolates were identified as GBS depending on cultural and microscopical properties, automated (VITEK-2 system), andmolecular identification based on atr gene, which is an essential gene expressed isolates in all S.agalactiae. Polymerase chain reaction(PCR) was used to detect the prevalence of virulence factors and antibiotic resistance genes; ermB gene was found in 22/22(100%),hly gene in twenty-one isolates (95.45%), scpB also found in twenty-one isolates (95.45%), tet gene (95.45%), mefA gene12/22(54.54%), Rib gene was found only in five isolates (22.72%), bca found in three isolates only (13.63%), bac not found in anyisolates(0%).

Study of the biofilm form of vibrio cholera by RT-PCR

BACKGROUND: Vibrio cholerae can exist in planktonic and biofilm forms. There are no unified methods for recording the formation of a biofilm and the quantitative determination of microorganisms; the known methods are laborious and do not allow an objective assessment of the concentration of V. cholerae in biofilms.&#x0D; AIM: This study aimed to evaluate the method for the quantitative determination of V. cholerae in biofilm and planktonic forms based on real-time PCR.&#x0D; MATERIALS AND METHODS: The concentration of V. cholerae in plankton was determined by the bacteriological method based on the number of colony-forming units per 1 ml; in biofilms, the method of imprint depletion on agar plates was used. Real-time PCR was performed using the primers and probes described in the literature for detecting the hlyA and ctx genes. V. cholerae were quantified using built-in software and standard preparations with a known concentration of bacterial cells. The results were processed in Microsoft Office Excel 2016 spreadsheets using the decimal logarithm; statistical analysis was performed using the Statistica 13.3 program.&#x0D; RESULTS: During the observation period, the concentration of V. cholerae in biofilms on chitin and plastic increases as the incubation period increases. The amount of V. cholerae in the composition of biofilms and plankton on/above chitin exceeded those when used as a plastic substrate. On the 30th day, the difference was two or more orders of magnitude. The results of the two methods were reproducible and comparable; at the same stages, the concentration of V. cholerae varied within the same order of magnitude, which indicated the reliability of the PCR-RT results.&#x0D; CONCLUSION: The bacteriological method is informative in the qualitative assessment of biofilms, in determining the viability of cholera vibrios. However, due to its complexity, with the impossibility of quickly determining the concentration of V. cholerae in a biofilm on chitin, it is preferable to use real-time PCR, which allows you to assess the concentration of V. cholerae in plankton and biofilm accurately and quickly.

A novel detection method for the pathogenic Aeromonas hydrophila expressing aerA gene and/or hlyA gene based on dualplex RAA and CRISPR/Cas12a.

Aeromonas hydrophila is an emerging waterborne and foodborne pathogen with pathogenicity to humans and warm water fishes, which severely threatens human health, food safety and aquaculture. A novel method for the rapid, accurate, and sensitive detection of pathogenic A. hydrophila is still needed to reduce the impact on human health and aquaculture. In this work, we developed a rapid, accurate, sensitive, and visual detection method (dRAA-CRISPR/Cas12a), without elaborate instruments, integrating the dualplex recombinase-assisted amplification (dRAA) assay and CRISPR/Cas12a system to detect pathogenic A. hydrophila expressing aerA and/or hlyA virulence genes. The dRAA-CRISPR/Cas12a method has high sensitivity, which can rapidly detect (about 45 min) A. hydrophila with the limit of detection in 2 copies of genomic DNA per reaction, and has high specificity for three pathogenic A. hydrophila strains (aerA+hlyA−, aerA−hlyA+, and aerA+hlyA+). Moreover, dRAA-CRISPR/Cas12a method shows satisfactory practicability in the analysis of the spiked human blood and stool and fish samples. These results demonstrate that our developed pathogenic A. hydrophila detection method, dRAA-CRISPR/Cas12a, is a promising potential method for the early diagnosis of human A. hydrophila infection and on-site detection of A. hydrophila in food and aquaculture.

Presence and virulence characterization of Listeria monocytogenes from fish samples in the Black Sea, Turkey

Listeria monocytogenes, characterized by a high mortality rate in humans, is a bacterium that causes listeriosis and is found in various aquatic products. The aim of this research was to investigate the presence, serotype distribution, virulence factor genes, and antibiotic susceptibility of L. monocytogenes strains isolated from a total of 500 fish samples of whiting (Merlangius merlangus euxinus) (n:243) and striped red mullet (Mullus surmuletus) (n:257) caught in the Black Sea between the years 2013-2014. Only one (0.2%) L. monocytogenes strain (striped red mullet) was isolated according to the cultural method (EN ISO 11290-1) and confirmed by PCR analysis. The L. monocytogenes strain was identified as serogroup 4b-4d-4e. Furthermore, the strain harboured hlyA, inlA, inlC, inlJ, plcA, plcB, prfA, mpl, actA, monoA-B, flaA, lip 1-2a, fri, iap, and gtcA genes except the dltA gene. On the other hand, L. monocytogenes strain susceptibility to ampicillin, meropenem, erythromycin, trimetophrim/sulfamethoxazole, and penicillin G was evaluated with the disc-diffusion method. According to the results, serogroup 4b-4d-4e isolated from striped red mullet was found to be unique to raw fish and susceptible to all tested antibiotics. In addition, it is considered that carrying out this research in different seas and with different fish species would be appropriate for determining the prevalence and virulence characteristics of L. monocytogenes.

Molecular identification of diarrheagenic Escherichia coli pathotypes and their antibiotic resistance patterns among diarrheic children and in contact calves in Bahir Dar city, Northwest Ethiopia.

Diarrheagenic Escherichia coli strains are an essential cause of diarrheal infection in younger children and animals. The study was focused on understanding the associated characteristics of various DEC strains among children and calves, establishing the possible zoonotic transmission, and determining their antibiotic resistance patterns. Samples from 144 acute diarrheic children and 50 diarrheic calves were collected and processed using traditional culture methods. The molecular identification of pathotypes was completed using primer-specific polymerase chain reaction (PCR) targeting ten virulence genes (stx1, stx2, eae, aatA, lt, st, ial, hlyA bfpA, and daaE) related to six DEC pathotypes (EPEC, ETEC, EHEC, EAEC EIEC, and DAEC). The antimicrobial susceptibility testing was carried out using the Kirby-Bauer disk diffusion method. Colonies from 74 study subjects (54 diarrheic children and 20 diarrheic calves) were positive for E. coli isolates. Subsequent PCR detection discovered that 77% of children and 85% of calves’ isolates were positive for one or more virulence genes typical of particular strains. Among those ETEC [(18%), (26%)] is being the maximum predominant, and [(15%), (15%)] were positive for STEC, [(13%), (8%)] for atypical EPEC, [(6%), (7%)] for EHEC, [(6%), (5%)] for EAEC, and [(6%), (4%)] for EIEC strains in children’s and calves, respectively. Of the identified E. coli isolates, about 29% were found to be hybrid isolates. ETEC (66.7%) and STEC (58.9%) strains showed a better detection rate in contact children with diarrheic calves than children with no contacts. Most antibiotic resistances were obtained towards amoxicillin (64.9%), gentamycin (56.8%), and ampicillin (54.1%). Up to sixty-five percent of isolates were resistant to a minimum of three categories of antibiotics. This is the primary report on the wide occurrence of the six-diarrheagenic Escherichia coli strains, and ETEC was found to be the predominant pathotype among children and contact calves in Ethiopia.

Molecular characterization and biofilm-formation analysis of Listeria monocytogenes, Salmonella spp., and Escherichia coli isolated from Brazilian swine slaughterhouses.

This study aimed to verify the presence of Listeria monocytogenes, Salmonella spp., and Escherichia coli in two Brazilian swine slaughterhouses, as well as to perform antibiograms, detect virulence and antimicrobial resistance genes, and evaluate the in vitro biofilm-forming capability of bacterial isolates from these environments. One Salmonella Typhi isolate and 21 E. coli isolates were detected, while L. monocytogenes was not detected. S. Typhi was isolated from the carcass cooling chamber’s floor, resistant to several antimicrobials, including nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline, and sulfonamide, and contained resistance genes, such as tet(B), tet(C), tet(M), and ampC. It also showed moderate biofilm-forming capacity at 37°C after incubating for 72 h. The prevalence of the 21 E. coli isolates was also the highest on the carcass cooling chamber floor (three of the four samplings [75%]). The E. coli isolates were resistant to 12 of the 13 tested antimicrobials, and none showed sensitivity to chloramphenicol, an antimicrobial prohibited in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), and blaSHV, as well as the virulence genes stx-1, hlyA, eae, tir α, tir β, tir γ, and saa were detected in the E. coli isolates. Moreover, 5 (23.8%) and 15 (71.4%) E. coli isolates presented strong and moderate biofilm-forming capacity, respectively. In general, the biofilm-forming capacity increased after incubating for 72 h at 10°C. The biofilm-forming capacity was the lowest after incubating for 24 h at 37°C. Due to the presence of resistance and virulence genes, multi-antimicrobial resistance, and biofilm-forming capacity, the results of this study suggest a risk to the public health as these pathogens are associated with foodborne diseases, which emphasizes the hazard of resistance gene propagation in the environment.