Determination of antibiotic resistance genes and virulence factors in Escherichia coli isolated from Turkish patients with urinary tract infection

Abstract INTRODUCTION : Escherichia coli ranks among the most common sources of urinary tract infections (UTI). METHODS: Between November 2015 and August 2016, 90 isolates of E. coli were isolated from patients at Rize Education and Research Hospital in Turkey. Antibiotic susceptibility was determined for all isolates using the Kirby-Bauer disk diffusion method. These E. coli isolates were also screened for virulence genes, β-lactamase coding genes, quinolone resistance genes, and class 1 integrons by PCR. RESULTS: With respect to the antibiotic resistance profile, imipenem and meropenem were effective against 98% and 90% of isolates, respectively. A high percentage of the isolates showed resistance against β lactam/β lactamase inhibitor combinations, quinolones, and cephalosporins. PCR results revealed that 63% (57/90) of the strains carried class 1 integrons. In addition, a high predominance of extended-spectrum β-lactamases (ESBLs) was observed. The qnrA, qnrB, and qnrS genes were found in 24 (26.6%), 6 (6.6%), and 3 (3.3%), isolates, respectively. The most common virulence gene was fim (82.2%).The afa, hly, and cnf1 genes were detected in 16.6%, 16.6%, and 3.3% of isolates, respectively. Moreover, we observed eleven different virulence patterns in the 90 E. coli isolates. The most prevalent pattern was fım, while hly-fım, afa-aer-cnf-fım, aer-cnf, afa-aer, and afa-cnf-fım patterns were less common. CONCLUSIONS: Most of the E. coli virulence genes investigated in this study were observed in E. coli isolates from UTI patients. Virulence genes are very important for the establishment and maintenance of infection.

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and alpha-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.

Characterization of Shiga-toxigenic Escherichia coli isolated from cases of diarrhoea & haemolytic uremic syndrome in north India.

Background & objectives: Shiga toxin producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen, capable of causing haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). As data from India on human infections caused by STEC are limited, this study was carried out for hospital based surveillance for STEC as a causative agent of diarrhoea, bloody diarrhoea and HUS at a tertiary care centre and to study the virulence gene profile and strain relatedness by multi locus variable tandem repeat analysis (MLVA). Methods: A total of 600 stool samples were studied. Stool samples of every fifth patient presenting with non-bloody diarrhoea, all cases of bloody diarrhoea and diarrhoea associated HUS (D+HUS) were collected from October 2009 to September 2011. Stool samples were cultured for STEC and characterization of STEC was done by serogrouping, virulence genes analysis, and MLVA typing. Results: STEC were isolated as a sole pathogen from 11 stool samples [5 of 290 (1.7%) non-blood diarrhoea and 5 of 300 (1.6%) blood diarrhoea cases]. STEC was also isolated from one fatal case of HUS who was an eight month old child. Only six of 11 isolates were positive for stx2 gene, whereas stx1 was present in all 11 isolates. Only one isolate was positive for eae. Other adhesion genes present were iha in five isolates, followed by toxB and efa1 in two each and saa gene in one, isolate. Among the plasmid encoded genes, espP, hly and etpD were each present in one isolate each. In the MLVA typing, diverse profiles were obtained except two untypeable isolates from different patients shared the same MLVA profile. Both these isolates were not epidemiologically linked. Interpretation & conclusions: This study demonstrated that STEC could be a causative agent of diarrhoea, bloody diarrhoea and sporadic HUS. However, further work needs to be done to study and explore the prevalence of these organisms in the food chain in this region.

Occurrence and characteristics of virulence genes of Escherichia coli strains isolated from healthy dairy cows in Inner Mongolia, China

Virulence genes of Escherichia coli (E. coli) isolates from healthy dairy cows were identified and characterized by a multiplex PCR assay and serogrouping test. The results showed that among the target genes, eaeA was most frequently detected, accounting for 22.11% (67/303) in all strains from 101 cows. For categorization of E. coli, aEPEC was the category with widest distribution detected in 55 (18.15%) strains from 22 cattle. All of 84 PCR-positive strains belonged to 14 O serogroups, and O149 (25.00%) was most common identified, followed by O2 (17.86%), O8 (11.90%) and O103 (9.52%) with relatively high prevalence.

Clinical and bacteriologic correlates of the PapG alleles among uropathogenic escherichia coli strains isolated from cases of adult urinary tract infection

To study the distribution of papG gene in uropathogenic Escherichia coli [E.coli] strains isolated from adult urinary tract infection [UTI] and the relationship between the different classes of papG gene and patients, sex, hospitalization and their clinical forms of UTI. Laboratory study. Inpatient and outpatient settings with laboratory investigation. Genotyping of papG, the adhesion gene of E. coli P fimbriae, may predict clinical outcomes of UTI. A total of 182 urinary E .coli strains were analyzed by multiplex PCR method for detection of papG gene. Patients, sex, hospitalization and their clinical forms of UTI were also evaluated. The distribution of papG gene in uropathogenic E.coli strains and the relationship between papG gene and clinical features of the patients. Multiplex PCR method was performed for detection of papG gene in uropathogenic E.coli strains isolated from adult urinary tract infections The prevalence of pap operon in the uropathogenic isolates was 36.2%. The prevalence of papG gene classes II and III in uropathogenic isolates was 23.1% and 6.6% respectively. None of the isolates had class I genotype. PapG classes II and III were predominant in patients with pyelonephritis and cystitis respectively. There was no significant relationship between the presence of papG alleles, sex and hospitalization of the patients. PapG gene is likely to play an important role in pathogenesis of uropathogenic strains of E.coli in adult nosocomial UTIs. Detection and genotyping of this gene may contribute to improving the management of UTI

QRDR mutations, efflux system & antimicrobial resistance genes in enterotoxigenic Escherichia coli isolated from an outbreak of diarrhoea in Ahmedabad, India.

Background & objectives: Diverse mechanisms have been identified in enteric bacteria for their adaptation and survival against multiple classes of antimicrobial agents. Resistance of bacteria to the most effective fluoroquinolones have increasingly been reported in many countries. We have identified that most of the enterotoxigenic Escherichia coli (ETEC) were resistant to several antimicrobials in a diarrhoea outbreak at Ahmedabad during 2000. The present study was done to identify several genes responsible for antimicrobial resistance and mobile genetic elements in the ETEC strains. Methods: Seventeen ETEC strains isolated from diarrhoeal patients were included in this study. The antimicrobial resistance was confirmed by conventional disc diffusion method. PCR and DNA sequencing were performed for the identification of mutation in the quinolone resistance-determining regions (QRDRs). Efflux pump was tested by inhibiting the proton-motive force. DNA hybridization assay was made for the detection of integrase genes and the resistance gene cassettes were identified by direct sequencing of the PCR amplicons. Results: Majority of the ETEC had GyrA mutations at codons 83 and 87 and in ParC at codon 80. Six strains had an additional mutation in ParC at codon 108 and two had at position 84. Plasmid-borne qnr gene alleles that encode quinolone resistance were not detected but the newly described aac(6’)-Ib-cr gene encoding a fluoroquinolne-modifying enzyme was detected in 64.7 per cent of the ETEC. Class 1 (intI1) and class 2 (intI2) integrons were detected in six (35.3%) and three (17.6%) strains, respectively. Four strains (23.5%) had both the classes of integrons. Sequence analysis revealed presence of dfrA17, aadA1, aadA5 in class 1, and dfrA1, sat1, aadA1 in class 2 integrons. In addition, the other resistance genes such as tet gene alleles (94.1%), catAI (70.6%), strA (58.8%), blaTEM-1(35.2%), and aphA1-Ia (29.4%) were detected in most of the strains. Interpretation & conclusions: Innate gene mutations and acquisition of multidrug resistance genes through mobile genetic elements might have contributed to the emergence of multidrug resistance (MDR) in ETEC. This study reinforces the necessity of utilizing molecular techniques in the epidemiological studies to understand the nature of resistance responsible for antimicrobial resistance in different species of pathogenic bacteria.

Molecular detection and epidemiology of Extended-Spectrum beta-lactamase genes prevalent in clinical isolates of Klebsiella pneumoniae and E coli from Trinidad and Tobago / Detección molecular y epidemiología de genes de beta-lactamasas de Espectro extendido prevalecientes en los aislados clínicos de Klebsiella pneumoniae y E coli en Trinidad y Tobago

OBJECTIVE: The epidemiology of Extended-spectrum beta-lactamase (ESBL) producing E coli and K pneumoniae is complex and varies among hospitals and countries. This study aimed at describing the molecular detection and epidemiology of ESBL subtypes prevalent in clinical isolates of K pneumonia and E coli in Trinidad and Tobago. METHODS: Over 36-months, isolates of E coli and K pneumoniae from clinical specimens of patients processed at a regional tertiary hospital in the country were identified using standard microbiological methods. MicroScan System (Siemens, USA) was used to determine MIC values while E-test (AB Biodisk, Sweden) assays phenotypically confirmed ESBL production. K pneumoniae (n= 65) and E coli (n = 25) isolates confirmed as ESBL producers were further subjected to multiplex PCR and PFGE tests to determine the ESBL subtypes and clonal relatedness. RESULTS: Female patients (67.8%) and urine samples (65%) yielded most ESBL isolates, with over 90% recovered from the hospital s medicine and surgery facilities. All ESBL isolates including all K pneumoniae producing ESBLs were 100% susceptible to carbapenems and amikacin antimicrobials. Polymerase Chain Reaction detected 100% blaTEM genes, 4.1% blaSHV and 37.5% bla cTX-M genes among E coli isolates. Similarly, 84.3% blaTEM, 34.5% blaSHv and 58.8% bla cTX-M genes were detected in K pneumoniae. Pulsed-field gel electrophoresis (PFGE) results showed diverse and unrelated clones. CONCLUSIONS: In this the first report of molecular characterization and epidemiology of ESBL subtypes in E coli and K pneumoniae isolates in Trinidad and Tobago, the CTX-M, mainly phylogenetically group 1 type, was most predominant. Most ESBL isolates were still susceptible to carbapenems and aminoglycosides and their spread appears to be polyclonal and clonally unrelated.

OBJETIVO: La epidemiología de E coli y K pneumoniae productores de beta-lactamasas de espectro extendido (BLEE) es compleja y varía de un hospital o país a otro. Este estudio tiene por objeto describir la detección molecular y la epidemiología de los subtipos de BLEE prevalecientes en aislados clínicos de K pneumonia y E coli en Trinidad y Tobago. MÉTODOS: Por más de 36-meses, se identificaron aislados de E coli y K pneumoniae a partir de especímenes clínicos de pacientes procesados en el hospital universitario regional del país, utilizando métodos microbiológicos estándar. Se utilizó el sistema MicroScan (Siemens, USA) para determinar los valores MIC, en tanto que la prueba del epsilómetro o E-test (biodisco AB, Suecia) confirmó fenotípicamente la producción de BLEE. Los aislados de K pneumoniae (n = 65) y E coli (n = 25) confirmados como productores de BLEE, fueron posteriormente sometidos a pruebas PCR multiplex y PFGE con el propósito de determinar los subtipos BLEE y la relación clonal. RESULTADOS: Las pacientes (67.8%) y las muestras de orinas (65%) arrojaron el mayor número de aislados de BLEE, siendo más del 90% tomados de las instalaciones de medicina y cirugía del hospital. Todos los aislados de BLEE, incluyendo todas las K pneumoniae productoras de BLEE resultaron 100% susceptibles a los agentes antimicrobianos carbapenem y amikacina. La reacción en cadena de la polimerasa detectó 100% de genes blaTEM, 4.1% de genes blaSHV y 37.5% de genes blaCTX-M entre los aislados de E coli. De manera similar, 84.3% de genes blaTEM, 34.5% blaSHV y 58.8% blaCTX-M fueron detectados en K pneumoniae. Los resultados de la electroforesis en gel de campos pulsados (PFGE) mostraron clones diversos y no relacionados. CONCLUSIONES: En este primer reporte de caracterización molecular y epidemiología de subtipos de BLEE en aislados de E coli y K pneumoniae en Trinidad y Tobago, el tipo principalmente filogenéticamente CTX-M grupo 1 fue el de mayor prevalencia. La mayoría de los aislados de BLEE eran todavía susceptibles a los carbapenems y los aminoglucósidos, y su extensión parece ser policlonal y no hallarse clonalmente relacionada.

Caracterização de Escherichia coli produtoras de toxina de Shiga (STEC) isoladas na produção de bovinos de corte e nas respectivas carcaças dos animais abatidos / Characterization of Shiga toxin-producing Escherichia coli (STEC) isolated in feedlot beef cattle production and their carcasses

Escherichia coli produtoras de toxina de Shiga (STEC) são considerados importantes patógenos de origem alimentar que apresentam o trato intestinal de ruminantes domésticos, principalmente bovinos, seu reservatório natural. Esses microrganismos estão associados com doenças severas em humanos, tais como colite hemorrágica (CH) e síndrome urêmica hemolítica (SHU). Este trabalho teve como objetivos avaliar a ocorrência de STEC em diferentes fontes, ambientais ou não, da criação e abate de bovinos confinados. Além disso, detectar a presença dos genes stx1, stx2, ehxA e eaeA; identificar cepas O157:H7 através da pesquisa do gene uidA; evidenciar a capacidade de produção de Stx e de Eh; identificar variantes de stx e de eaeA; e determinar os sorotipos a diversidade genética das cepas de STEC. A avaliação da presença dos genes (stx1, stx2, ehxA e eaeA) e da produção de Eh foi utilizada como triagem para a seleção de cepas possivelmente patogênicas, sendo que do total de 628 isolados avaliados, foram selecionadas 47 cepas STEC típicas e outras 12 consideradas como atípicas. Das STEC típicas 80,9% foram isolados provenientes de amostras de fezes, enquanto 19,1% foram de amostras de carcaças. Seis cepas isoladas de fezes e 1 de carcaça foram sorotipificadas como 0157:H7, todas positivas para a presença do gene uidA. Além do sorogrupo 0157, nenhum outro, dentre os principais causadores de surtos e casos esporádicos de CH e SHU, foi detectado...

Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections

Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4 per cent) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6 per cent). The pap operon was detected in 39 (53.4 per cent) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0 per cent) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8 per cent) of the urinary strains and in 16 (66.6 per cent) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5 per cent), cystitis (45.8 per cent) and asymptomatic bacteriuria (54.5 per cent). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100 per cent in pyelonephritis and in asymptomatic bacteriuria, and 87.5 per cent in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11-to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.