Virulence Genes iutA Research Articles

The in vitro Antimicrobial Effects of Hydrolysable and Condensed Tannin Extracts on Escherichia coli Isolated from Pathological Samples of Deceased Poultry

Abstract Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is one of the most prevalent and economically damaging bacterial diseases affecting poultry globally. Managing colibacillosis is difficult and frequently ineffective because APEC strains have developed widespread resistance to antibiotics, and the strict regulations and public concerns towards using antimicrobial agents in poultry further complicate the situation. This study aimed to evaluate the antibacterial properties of hydrolysable (sweet chestnut extract) and condensed (quebracho extract) tannins on Escherichia coli (E. coli) isolates from poultry, exploring their potential as antibiotic alternatives in managing colibacillosis. E. coli was isolated from the internal organs of deceased poultry across 18 farms, including layers, broilers, and broiler breeders. Each isolate was assessed for the presence of APEC strain predictors (virulence genes iutA, hlyF, iss, iroN, and ompT), antimicrobial resistance to 14 antibiotics using the disc diffusion method, and the presence of resistance genes for specific antibiotics (ampicillin, gentamicin, tetracycline, and quinolones). Out of 43 isolates, 27 (62.8%) were classified as APEC, 30 (69.8%) showed resistance to three or more antibiotic classes, and 32 (74.4%) carried at least one AMR gene. The minimum inhibitory concentrations (MICs) for the hydrolysable tannins from sweet chestnut extract (Castanea sativa Mill.) (SwCh) ranged from 0.5 to 3 mg/mL, while for the condensed tannins from quebracho extract (Schinopsis lorentzii) (Que), the MICs ranged from 1.5 to 4.5 mg/mL. The results indicate that both hydrolysable and condensed tannins possess significant in vitro antimicrobial activity against APEC, offering a potentially valuable alternative for controlling colibacillosis in the poultry industry.

Study of the relationship between antibiotic resistance markers and virulence markers in NDM-positive Klebsiella pneumoniae strains circulating in various waters and human loci

Introduction. The propagation of multi-resistance to antibiotics among hospital isolates of Klebsiella pneumoniae (K. pneumoniae) is a subject of growing concern worldwide. At present, growing data of association between resistance and hypervirulence in clinical isolates of K. pneumoniae emerges. However, the occurrence of these pathogens in the environment remains an open question. The aim of this study was to evaluate and compare antibiotic resistance determinants occurrence in Klebsiella pneumoniae isolates from water sources (environmental and sewage), human sources (practically healthy people and patients with inflaammatory bowel disease (IBD), and extraintestinal infections (ExII)). Materials and methods. The PCR assay of carbapenemase genes IMP, NDM, VIM, KPC, OXA-48 was performed with the commercial “Amplisense” kits according to the manufacturer's instructions. The assay was used to evaluate the occurrence of antibiotic-resistance genes in 223 isolates of Klebsiella pneumoniae from various sources: 42 isolates from sewage, 19 isolates from surface water sources, 30 isolates from biological material (blood, urine, surgical wounds, bronchoalveolar lavage) of patients with extraintestinal infections (ExII), 69 isolates from patients with inflammatory bowel diseases (IBD), and 63 isolates from faeces of practically healthy people. Results. The ExII group revealed various antibiotic resistance genes. The most prevalent gene was OXA (30% had this gene only, other 26,6% had also KPC or NDM). NDM as the only resistance gene was observed in 23,3% of ExII isolates. KPC gene was observed in 3,3% of ExII group. Two isolates from IBD group contained NDM gene along with VIM gene. Only NDM gene was found in all the other groups of Klebsiella pneumoniae isolates (13-28% isolates in every group, no statistical difference). NDM was shown to be associated with virulence genes iutA and rmpA that are responsible for iron consumption and hypermucoid phenotype. Conclusion. The most abundant resistance genes in the studied Klebsiella pneumoniae isolates were NDM (13.5%) and OXA (8%). At the same time, NDM was the only gene found in all groups (11-28%). NDM metallobeta-lactamase gene was associated with rmpA and iutA genes, giving an example of the connection between virulence and resistance properties. A significant amount of resistant isolates from healthy donors and surface waters indicates the need for additional study of the role of NDM positive isolates in pathogenicity of Klebsiella pneumoniae.

Characterization of a multidrug-resistant Klebsiella pneumoniae ST3330 clone responsible for a nosocomial outbreak in a neonatal intensive care unit.

The incidence of Klebsiella pneumonia (Kp), which has often been found to produce, extended spectrum beta-lactamase (ESBL), is rising rapidly and poses a serious risk to neonates. To date, the mechanisms related to the spread of ESBL-Kp have not been fully elucidated. This study aimed to investigate the phenotypes, genotypes, and genetic relatedness of ESBL-KP that caused an outbreak of sepsis among neonates in an intensive care unit of a Beijing hospital. Between April 2016 and May 2018, 21 non-repetitive clinical ESBL-Kp isolates were collected from a neonatal intensive care unit (NICU) in Beijing, China and were retrospectively analyzed. Pulsed-field gel electrophoresis (PFGE) was used to analyze genetic relatedness, a VITEK 2 AST test kit was used to test antimicrobial susceptibility, sequence type (ST) was analyzed through multilocus sequence typing (MLST), and resistance genes were identified by PCR. Virulence gene profiles, biofilm formation assay, and serum killing assay were used for virulence-associated determinants. All strains expressed the same antibiotype, combining ESBL production, third generation cephalosporins resistance and carbapenems sensitive. Sixteen of them produced β-lactamases (CTX-M-3 and TEM-1B), while others possessed CTX-M-15, CTX-M-24, CTX-M-66, TEM-1C, SHV-26, SHV172, and OXA-1. PFGE confirmed 5 types (A, B, C, D and E) and MLST identified a ST3330 clone (16 strains), a ST2791 clone (2 strains), a ST37 clone (1 strain), a ST34 clone (1 strain), and a ST2740 clone (1 strain). PFGE type A strains, which belong to ST3330, were identified as the main pathogens involved in the outbreak. All isolates contained virulence genes iutA and mrk. PFGE type A carried both mrk (type 3 fimbriae, biofilm formation) and fimH (type 1 fimbriae), and other STs possessed mrk. Isolates belonging to the endemic ST3330 lineage produced more biofilm than other ST isolates (median OD590 1.829 vs. 0.2280, respectively; P<0.0001). All five PFGE types isolates showed serum high sensitivity (grade 1). The dissemination and outbreak of ESBL-producing K. pneumoniae in this study seemed to be clonal, and the outbreak was mainly caused by ST3330 K. pneumoniae. The detection of genes (mrk and fimH) belonging to the biofilm formation may partly explain the epidemic strain has high colonization and diffusion potential.

The relation of phylogroups, serogroups, virulence factors and resistance pattern of Escherichia coli isolated from children with septicemia.

The characterization of virulent and drug-resistant Escherichia coli strains helps to control and provide more accurate information regarding infection and eradication. The aim of this study was to determine the relationship between antibiotic susceptibility, phylogroups and virulence factors of E. coli isolates from children with septicaemia. One hundred dereplicated E. coli isolates were collected from paediatric patients with septicaemia in five hospitals in Tehran (May 2015 to May 2018). The antibiotic susceptibility of isolates was performed as per the 2016 guidelines of the Clinical and Laboratory Standards Institute. Extended-spectrum β-lactamases and carbapenemase genes, phylogroups, serogroups and virulence encoding genes were detected by PCR. Phylogroup B2 was dominant (40%) among strains, followed by phylogroups D (30%), A (8%) and B1 (7%). CTX-M1 was significantly higher in the B2 group (n = 21, p 0.001). Furthermore, the virulence genes iutA (n = 27, p 0.002), csgA (n = 39, p <0.001), kpsMII (n = 39, p 0.002), ibeA (n = 4, p 0.004), vat (n = 5, p 0.003), traT (n = 24, p <0.001), sat (n = 12, p 0.001) and hlyA (n = 33, p <0.001) showed significantly higher rates in phylogroup B2. Three O25/CTXM1/OXA-48 and cnf, iutA, csgA and traT positive isolates belonged to phylogroup B2. Pulsed-field gel electrophoresis analysis showed 85% similarity among 25% of isolates. More than half of the isolates were multidrug-resistant E. coli. A significant relation was observed among iutA, csgA, kpsMII, ibeA, vat, traT, sat and hlyA genes and phylogroup B2. The characterization of virulent and drug-resistant strains helps control and properly eliminate infections. There was no genetic relation among strains in the pulsed-field gel electrophoresis pattern.

Evaluation of bulk tank raw milk and raw chicken meat samples as source of ESBL producing Escherichia coli in Turkey: Recent insights

AbstractExtended spectrum β‐lactamase producing Escherichia coli (ESBL‐EC) was detected in 86.6% of chicken and 22.6% of bulk tank milk (BTM) samples. Pulsed‐field gel electrophoresis analysis revealed 49 distinct restriction profiles among 66 isolates, and 62.3% of the isolates carried the blaCTX‐M gene, among which CTX‐M‐1 was found to be the predominant ESBL types in chicken isolates, whereas CTX‐M‐15 was the commonest among BTM samples. Additionally, of 52 ESBL‐EC isolates from chicken meat samples, 36.5%, 9.6%, and 7.7% harbored the blaTEM, blaCMY‐2, and blaSHV‐12 genes, respectively, compared with 28.5% of the blaTEM and 7.1% of the blaSHV‐12 markers among BTM isolates. The fimH gene was present in 51 isolates of chicken and in 14 isolates of BTM samples, while other virulence genes iutA (n = 31), iroN (n = 26), kpsMT II (n = 5), papC (n = 2), papG allele II (n = 2), papG allele II‐III (n = 2), and papEF (n = 2) were only present in chicken meat isolates. Overall, it can be said that contaminated chicken meat and BTM might serve as vehicles for playing potential role in zoonotic transmission of ESBL‐EC to humans in Turkey.Practical applicationsExtended spectrum β‐lactamase producing E. coli (ESBL‐EC) have been considered to be one of the major worldwide clinical problems and have been frequently isolated from the foods of animal origins such as chicken meat, fish, and raw milk. The current study aimed to isolate ESBL‐EC from chicken and bulk tank milk samples and further characterize the strains by PFGE, identifying resistance genes and as well as virulence genes. Given the high prevalence of CTX‐M‐15/55 type ESBL‐EC strains possessing important virulence genes in chicken and milk samples, it can be said that foods of animal origins might be an important risk factor for extraintestinal ESBL‐EC infections for humans.

Complete and assembled genome sequence of an NDM-5- and CTX-M-15-producing Escherichia coli sequence type 617 isolated from wastewater in Switzerland.

Carbapenem-resistant Escherichia coli have emerged worldwide and represent a major challenge to effective healthcare management. Here we report the genome sequence of an NDM-5- and CTX-M-15-producing E. coli belonging to sequence type 617 isolated from wastewater treatment plant effluent in Switzerland. Whole-genome sequencing of E. coli 657SK2 was performed using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology RS2 reads (C4/P6 chemistry). De novo assembly was carried out using Canu 1.6, and sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The genome of E. coli 657SK2 consists of a 4.9-Mbp chromosome containing blaCTX-M-15, genes associated with virulence [fyuA, hlyE, the pyelonephritis-associated pili (pap) gene cluster and the yad gene cluster], the copper resistance gene pco, and genes associated with resistance to quaternary ammonium compound (QAC) disinfectants (emrA, mdfA and sugE). A 173.9-kb multidrug resistance IncFII-FIA-FIB plasmid was detected harbouring aadA2, aadA5, blaNDM-5, blaOXA-1, cat, drfA, drfA17, the mph(A)-mrx-mphR cluster, the tetA-tetC-tetR cluster, and the virulence genes iutA and ylpA. The genome sequence of E. coli 657SK2 provides information on resistance mechanisms and virulence characteristics of pathogenic E. coli harbouring blaNDM-5 and blaCTX-M-15 that are spreading into the environment via urban wastewater.

Occurrence of bla CTX-M-1, qnrB1 and virulence genes in avian ESBL-producing Escherichia coli isolates from Tunisia.

Avian ESBL-producing Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored blaCTX-M-1 gene, and one strain co-harbored the blaTEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.

Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis

Avian pathogenic Escherichia coli, the causative agent of colibacillosis, harbors several putative virulence genes. In this study we examined by polymerase chain reaction (PCR) the presence of 16 of those genes in 200 colibacillosis isolates from our region. The seven virulence genes iutA, iss, cvaC, tsh, papC, papG and felA were detected significantly more often amongst colibacillosis isolates than in fecal isolates from healthy birds, thereby confirming their worldwide occurrence and possible pathogenic role in colibacillosis. However, several of those genes were not detected in many colibacillosis isolates, and none of them were detected in 27.5% of those isolates, which suggests that variants of those genes and yet undetected virulence factors should be searched for.