Common Extended-spectrum Beta-lactamase Research Articles

Coexistence of a nonresistance-conferring IncI1 plasmid favors persistence of the blaCTX-M-bearing IncFII plasmid in Escherichia coli.

So far, plasmid-carried blaCTX-M is still the most common extended-spectrum beta-lactamase (ESBL) genotype in clinical settings worldwide. Except for the widespread use of third-generation cephalosporins, the interaction between coexisting plasmids can also affect the long-term stable existence of the blaCTX-M gene; however, the study on that is still sparse. In the present study, we assess the interaction of coinhabitant plasmids blaCTX-M-IncFII and nonresistance-IncI1. Our results confirmed that the increased fitness advantages of strain C600FII+I1 were attributable to the cohabitant nonresistance-IncI1 plasmid, which largely reduced the intracellular ATP levels of host bacteria, thus decreasing the rep gene expression of the blaCTX-M-IncFII plasmid, its copy numbers, and mating efficiencies, while the higher fitness advantages of strain C600FII+I1 enhanced the persistence of the blaCTX-M-IncFII plasmid. The results indicate that the nonresistance-IncI1 plasmid contributes to the long-term existence of the blaCTX-M-IncFII plasmid, implying a potentially new strategy for controlling the spread of resistance plasmids in clinical settings by targeting nonresistance plasmids.

Prevalence of Extended-Spectrum β-Lactamase-Resistant Genes in Escherichia coli Isolates from Central China during 2016-2019.

The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016-2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/μL, 1.10 × 101 copies/μL, and 1.86 × 101 copies/μL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.

Isolation, Identification, Characterization, and Plasmid Profile of Urinary Tract Infectious Escherichia coli from Clinical Samples.

Objective In recent times, urinary tract infection (UTI) is one of the most widely recognized bacterial diseases all over the planet. UTI influences individuals of any age and gender. The target of this study is to concentrate on the recurrence of uropathogens, the antimicrobial susceptibility pattern of the isolates, and the plasmid profile of people from the government clinics of Karaikudi. Methods From July 2017 to December 2017, 100 urine tests were gathered and handled for the isolation of pathogenic microbes. In total, 89 isolates were found from the samples collected. Results Escherichia coli was discovered as the most common bacterial isolate screened from the UTI-infected people, accounting for 28.09 percent of all isolates. E. coli was seen to be the highest prevalent bacterium for UTI in all age groups and demonstrated resistance to routinely used medications, especially cefpodoxime and novobiocin, which have been 100 percent resistant. The E. coli isolates screened were positive for beta-lactamase and film generation, and they have strong antimicrobial resistance. As a result, the E. coli strains with the highest prevalence of virulence determinants have become more resistant to many medications because they support the microorganism in overcoming the host's defense and colonizing or entering the urinary system. The amplified 16S rRNA product was analyzed, and phylogenetic relationships were determined. The presence of TEM (56 percent), CTX-M (64 percent), SHV (40 percent), and OXA (60 percent) was discovered. Among E. coli isolates, CTX-M was the most common extended spectrum-beta lactamase (ESBL). Multiplex PCR was also used to identify the existence of CTX-M subgroups in E. coli isolates. Conclusion Finally, we urge that antibiotic selection should be predicated on the awareness of the specific prevalence and that novel antimicrobial medicines for urinary infections be developed to combat the overuse of antibiotics.

Effect of the Different Plasmid-Mediated AmpC Beta-Lactamase Genotypes on the Phenotypic Detection of ESBL in Enterobacteriaceae Isolates.

Increasingly, Enterobacteriaceae isolates that are positive for the extended-spectrum-beta-lactamase (ESBL) by phenotypic screening tests yield a negative result when tested with the reference Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The aim is to determine to what extent the different plasmid AmpC (pAmpC) genotypes could affect the CLSI ESBL confirmatory test for detection of the ESBL phenotype in ESBL/pAmpC co-producers. A total of 253 Enterobacteriaceae isolates were screened for ESBL and AmpC production according to CLSI guidelines. Out of 186 ESBL and AmpC-screen-positive isolates, 96 isolates were selected for ESBL confirmation by the combined disc diffusion test (CDDT) as well as for detection of the most common ESBL and pAmpC encoding-genes by multiplex PCR. Out of the 96 ESBL/AmpC-screen-positive isolates, all (100%) were positive for at least one of the investigated ESBL genes, and 88 (91.7%) were positive for any of the investigated pAmpC genes. CDDT correctly identified ESBL phenotype more frequently in non-pAmpC carriers than pAmpC carriers (75% vs. 52.3%). CIT alone-containing isolates were associated more with non-confirmed ESBL phenotype rather than confirmed ESBL phenotype (76.2% vs. 30.4%, p < 0.001), especially when co-harbored blaCTXM alone (76.9% vs. 33.3%, p < 0.001) or both blaCTXM/blaSHV genes (100% vs. 0%). On the other hand, DHA-carrying isolates were more associated with confirmed ESBL phenotype than with non-confirmed ESBL phenotype when co-harboring either blaCTXM alone (47.6% vs. 0%, p < 0.001) or blaCTXM/blaSHV genes (100% vs. 0%, p = 0.022) or blaCTX-M/blaTEM genes (100% vs. 0%, p = 0.03). For ESBL/pAmpC co-producers of Enterobacteriaceae, CDDT results vary with the type of pAmpC genes and with different ESBL/pAmpC genotype combinations. Therefore, the ESBL-screening test is more sensitive than the CDDT in detecting ESBL phenotype among ESBL/pAmpC coproducers of Enterobacteriaceae.

743. Colonization with Extended-Spectrum Cephalosporin-Resistant Enterobacterales (ESCrE) in Healthcare and Community Settings in Botswana: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study

Abstract Background Although ESCrE are a global challenge, data on ESCrE in low- and middle-income countries are limited. In particular, colonization data are critical for larger antibiotic resistance efforts. We characterized the colonization prevalence of ESCrE in various settings in Botswana. Methods This study was conducted in 3 hospitals and 6 clinics located in 3 districts in Botswana. In each hospital, we conducted surveillance of adult patients. Adult clinic patients were also randomly selected for participation. Finally, we enrolled community subjects by inviting each enrolled clinic subject to refer up to 3 adults. Each adult clinic or community subject was also allowed to refer their children. All subjects had rectal swabs obtained which were inoculated onto chromogenic media for preliminary identification of ESCrE. Final identification and susceptibility testing were performed using MALDI-TOF MS and VITEK-2, respectively. Genotyping was done for identification of extended-spectrum beta-lactamase (ESBL) genes. Results Enrollment occurred from 1/15/20-9/4/20 but paused from 4/2/20-5/21/20 due to a countrywide COVID lockdown. Of 5,088 subjects approached, 2,469 (49%) participated. Enrollment by subject type was: hospital – 469 (19%); clinic – 959 (39%); community adult – 477 (19%); and community child – 564 (23%). Of 2,469 subjects, the median (interquartile range) age was 32 years (19-44) and 1,783 (72%) were female. 759 (31%) subjects were colonized with at least one ESCrE; 130 subjects were colonized with multiple strains. E. coli (n=663) and K. pneumoniae (n=121) were most common. ESCrE colonization prevalence was 43% for hospital subjects, 31% for clinic subjects, 24% for adult community subjects, and 26% for child community subjects (p&amp;lt; 0.001)). ESCrE prevalence varied significantly across regions (Figure 1) and was significantly higher pre-lockdown vs post-lockdown (Figure 2). CTX-M was the most common ESBL gene (Figure 3). Figure 1. ESCrE Colonization - Study Sites Figure 2. ESCrE Colonization - Temporal Trends Figure 3. ESCrE Genotypic Analyses Conclusion ESCrE colonization was common in both healthcare and community settings in Botswana. Colonization prevalence varies by region and clinical setting and decreased following a countrywide lockdown. These findings provide important clues regarding potential drivers of ESCrE that might serve as targets for intervention. Disclosures Robert Gross, MD, MSCE, Pfizer (Other Financial or Material Support, Serve on DSMB for drug unrelated to HIV) Ebbing Lautenbach, MD, MPH, MSCE, Merck (Other Financial or Material Support, Member of Data and Safety Monitoring Board (DSMB))

Molecular characterization of multiresistant enterobacteria in two departments of the Peruvian jungle

ResumenIntroducción. La aparición de enterobacterias multirresistentes y productoras de betalactamasas de espectro extendido (BLEE) en pacientes de consulta externa con infecciones urinarias, representa un problema de salud pública en Perú.Objetivos. Caracterizar molecularmente enterobacterias multirresistentes aisladas de pacientes con diagnóstico de infección urinaria y procedentes de dos departamentos de la selva peruana.Materiales y métodos. Se hizo un estudio descriptivo, observacional y retrospectivo de 61 aislamientos de urocultivo procedentes de la selva peruana durante 2017 y 2018. Los perfiles de resistencia se identificaron utilizando el sistema automatizado MicroScan™ y para la detección de los genes bla TEM , bla CTX-M , bla SHV se empleó una reacción en cadena de la polimerasa (PCR) convencional.Resultados. Las enterobacterias positivas para BLEE más frecuentes por departamento fueron Escherichia coli en Madre de Dios (25%, 10/40) y Ucayali (76,2%, 16/21). En ambos departamentos, el gen blaCTX-M fue el más frecuente (25/61; 41), seguido por bla TEM (15/61; 24,6%) y bla SHV (10/61; 16,4%). En el perfil de sensibilidad antimicrobiana, se detectó 72,6% de resistencia contra ampicilina, 82,3 % contra cefalotina y 88,7% contra nitrofurantoína.Conclusiones. El porcentaje de cepas de enterobacterias multirresistentes productoras de BLEE en ambos departamentos fue del 57,4% y el gen bla CTX-M fue el más frecuente.

Occurrence of Escherichia coli producing extended spectrum β-lactamases in food-producing animals.

Multidrug resistance due to the production of extended-spectrum beta-lactamases (ESBLs) is a major problem in human as well as in veterinary medicine. These strains appear in animal and human microbiomes and can be the source of infection both in animal and in human healthcare, in accordance with the One Health theorem. In this study we examined the prevalence of ESBL-producing bacteria in food-producing animals. We collected 100 porcine and 114 poultry samples to examine the prevalence of ESBL producers. Isolates were identified using the MALDI-TOF system and their antibiotic susceptibility was tested using the disk diffusion method. ESBL gene families and phylogroups were detected by polymerase chain reactions. The prevalence of ESBL producers was relatively high in both sample groups: 72 (72.0%) porcine and 39 (34.2%) poultry isolates were ESBL producers. Escherichia coli isolates were chosen for further investigations. The most common ESBL gene was CTX-M-1 (79.3%). Most of the isolates belong to the commensal E. coli phylogroups. The porcine isolates could be divided into three phylogroups, while the distribution of the poultry isolates was more varied. In summary, ESBL-producing bacteria are prevalent in the faecal samples of the examined food-producing animals, with a dominance of the CTX-M-1 group enzymes and commensal E. coli phylogroups.

Molecular detection of blaCTX-M gene to predict phenotypic cephalosporin resistance and clinical outcome of Escherichia coli bloodstream infections in Vietnam

BackgroundBlood stream infections (BSI) caused by Extended Spectrum Beta-Lactamases (ESBLs) producing Enterobacteriaceae is a clinical challenge leading to high mortality, especially in developing countries. In this study, we sought to describe the epidemiology of ESBL-producing Escherichia coli strains isolated from Vietnamese individuals with BSI, to investigate the concordance of genotypic-phenotypic resistance, and clinical outcome of ESBL E. coli BSI.MethodsA total of 459 hospitalized patients with BSI were screened between October 2014 and May 2016. 115 E. coli strains from 115 BSI patients were isolated and tested for antibiotic resistance using the VITEK®2 system. The ESBL phenotype was determined by double disk diffusion method following the guideline of Clinical and Laboratory Standards Institute. Screening for beta-lactamase (ESBL and carbapenemase) genes was performed using a multiplex-PCR assay.Results58% (67/115) of the E. coli strains were ESBL-producers and all were susceptible to both imipenem and meropenem. Resistance to third-generation cephalosporin was common, 70% (81/115) were cefotaxime-resistant and 45% (52/115) were ceftazidime-resistant. blaCTX-M was the most common ESBL gene detected (70%; 80/115) The sensitivity and specificity of blaCTX-M-detection to predict the ESBL phenotype was 87% (76–93% 95% CI) and 54% (39–48% 95% CI), respectively. 28%% (22/80) of blaCTX-M were classified as non-ESBL producers by phenotypic testing for ESBL production. The detection of blaCTX-M in ESBL-negative E. coli BSI was associated with fatal clinical outcome (27%; 6/22 versus 8%; 2/26, p = 0.07).ConclusionA high prevalence of ESBL-producing E. coli isolates harbouring blaCTX-M was observed in BSI patients in Vietnam. The genotypic detection of blaCTX-M may have added benefit in optimizing and guiding empirical antibiotic therapy of E. coli BSI to improve clinical outcome.

Antibiotic resistance patterns of Escherichia coli isolates from the clinic through the wastewater pathway.

Antimicrobial resistance (AMR) remains one of the leading global health threats. This study compared antimicrobial resistance patterns among E. coli isolates from clinical uropathogenic Escherichia coli (UPEC) to hospital wastewater populations and throughout an urban wastewater treatment facility - influent, pre- and post-chlorinated effluents. Antibiotic susceptibility of 201 isolates were analyzed against eleven different antibiotics, and the presence of twelve antibiotic resistant genes and type 1 integrase were identified. AMR exhibited the following pattern: UPEC (46.8%)>hospital wastewater (37.8%)>urban post-chlorinated effluent (27.6%)>pre-chlorinated effluent (21.4%)>urban influent wastewater (13.3%). However, multi-drug resistance against three or more antimicrobial classes was more prevalent among hospital wastewater populations (29.7%) compared to other sources. E. coli from wastewaters disinfected with chlorine were significantly correlated with increased trimethoprim-sulfamethoxazole resistance in E. coli compared to raw and treated wastewater populations. blaCTX-M-1 group was the most common extended spectrum beta-lactamase in E. coli from hospital wastewater (90%), although UPEC strains also encoded blaCTX-M-1 group (50%) and blaTEM (100%) genes. Among tetracycline-resistant populations, tetA and tetB were the only resistance genes identified throughout wastewater populations that were associated with increased phenotypic resistance. Further characterization of the E. coli populations identified phylogroup B2 predominating among clinical UPEC populations and correlated with the highest AMR, whereas the elevated rate of multi-drug resistance among hospital wastewater was mostly phylogroup A. Together, our findings highlight hospital wastewater as a rich source of AMR and multi-drug resistant bacterial populations.

Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa.

Wastewater treatment plants (WWTPs) are major reservoirs of antibiotic-resistant bacteria (ARB), favouring antibiotic resistance genes (ARGs) interchange among bacteria and they can provide valuable information on ARB circulating in a community. This study characterised extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli from the influent and effluent of four WWTPs in uMgungundlovu District, KwaZulu-Natal, South Africa. E. coli was enumerated using the membrane filtration method and confirmed using the API 20E test and real-time polymerase chain reaction. ESBL-producers were phenotypically identified by their susceptibility to the third-generation cephalosporins using the disc diffusion and the double-disc synergy methods against cefotaxime (30 µg) with and without 10 µg clavulanic acid. Genotypic verification was by PCR of the TEM, SHV, and CTX-M genes. The clonality of isolates was assessed by ERIC-PCR. The highest E. coli count ranged between 1.1 × 105 (influent) and 4.3 × 103 CFU/mL (effluent). Eighty pure isolates were randomly selected, ten from the influent and effluent of each of the four WWTP. ESBLs were phenotypically confirmed in 49% (n = 39) of the isolates, of which 77% (n = 30) were genotypically confirmed. Seventy-three percent of the total isolates were multidrug-resistant (MDR). Only two isolates were susceptible to all antibiotics. Overall, resistance to first and second-generation cephalosporins was higher than to third and fourth generation cephalosporins. Also, 15% of the isolates were resistant to carbapenems. The CTX-M-type ESBL (67%; n = 20) was the most common ESBL antibiotic resistance gene (ARG) followed by TEM (57%; n = 17) and SHV-types (27%; n = 8). Also, a substantial number of isolates simultaneously carried all three ESBL genes. ERIC-PCR revealed a high diversity of isolates. The diversity of the isolates observed in the influent samples suggest the potential circulation of different ESBL-producing strains within the studied district, requiring a more comprehensive epidemiological study to prevent the spread of ESBL-producing bacteria within impoverished communities.

Bloodstream infections in adult patients with malignancy, epidemiology, microbiology, and risk factors associated with mortality and multi-drug resistance

BackgroundThis study aimed to investigate the epidemiology, microbiology, and risk factors associated with mortality and multi-drug resistance bacterial bloodstream infections (BSIs) among adult cancer patients in Shiraz, Iran. We also report a four-year trend of antimicrobial resistance patterns of BSIs.MethodsWe conducted a retrospective study at a referral oncology hospital from July 2015 to August 2019, which included all adults with confirmed BSI.Results2393 blood cultures tested during the four-year study period; 414 positive cultures were included. The mean age of our patients was 47.57 ± 17.46 years old. Central Line-Associated BSI (CLABSI) was more common in solid tumors than patients with hematological malignancies. Gram-negative (GN) bacteria were more detected (63.3%, 262) than gram-positive bacteria (36.7%, 152). Escherichia coli was the most common gram-negative organism (123/262, 47%), followed by Pseudomonas spp. (82/262, 31%) and Klebsiella pneumoniae (38/262, 14.5%). Coagulase-negative staphylococci (CoNS) was the most frequently isolated pathogen among gram-positive bacteria (83/152, 54.6%). Acinetobacter spp., Pseudomonas spp., E. coli, and K. pneumoniae were the most common Extended-Spectrum Beta-Lactamase (ESBL) producers (100, 96.2, 66.7%, and 60.7, respectively). Acinetobacter spp., Pseudomonas spp., Enterobacter spp., E. coli, and K. pneumoniae were the most common carbapenem-resistant (CR) isolates (77.8, 70.7, 33.3, 24.4, and 13.2%, respectively). Out of 257 Enterobacterales and non-fermenter gram-negative BSIs, 39.3% (101/257) were carbapenem-resistant. Although the incidence of multi-drug resistance (MDR) gram-negative BSI increased annually during 2015–2018, the mortality rate of gram-negative BSI remains unchanged at about 20% (p-value = 0.55); however, the mortality rate was significantly greater (35.4%) in those with resistant gram-positive BSI (p-value = 0.001). The overall mortality rate was 21.5%. Early (7-day mortality) and late mortality rate (30-day mortality) were 10 and 3.4%, respectively.ConclusionsThe emergence of MDR gram-negative BSI is a significant healthcare problem in oncology centers. The high proportion of the most frequently isolated pathogens were CR and ESBL-producing Enterobacterales and Pseudomonas spp. We have few effective choices against MDRGN BSI, especially in high-risk cancer patients, which necessitate newer treatment options.

Multidrug resistance and extended-spectrum beta-lactamase producing Gram-negative bacteria from three Referral Hospitals of Amhara region, Ethiopia

BackgroundMultidrug resistance (MDR), extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing Gram-negative bacteria (GNB) has become a public health threat worldwide. This threat is worse in developing countries where there is high infectious disease burden and spread of antimicrobial resistance co-exist. The aim of the present study was, therefore, to assess MDR, ESBL and carbapenemase producing GNB from patients attending three selected referral hospitals in Amhara region.MethodsA cross-sectional study was conducted from December 2017- April 2018 at the University of Gondar Comprehensive Specialized Hospital, Dessie Referral Hospital and Debre Markos Referral Hospital of Amhara national regional state. A total of 833 study subjects were recruited using a convenient sampling technique. Clinical samples such as blood, urine, stool, wound, abscess, ear discharge, nasal discharge, cervical discharge and body fluid specimens were aseptically collected. Culturing for identification of bacteria and determination of drug susceptibility testing were done following standard microbiological techniques. Selected MDR isolates were phenotypically assessed for ESBL and carbapenemase production.ResultsOf the 833 clinical samples cultured for bacterial growth, 141 (16.9%) were positive for GNB. The most common GNB identified were E. coli 46 (32.6%), Klebsiella spp. 38 (26.5%) and Proteus spp. 13 (9.2%). The overall MDR prevalence was 121 (85.8%). Among the total isolates, 137 (97.2%) were resistant to ampicillin followed by cotrimoxazole 115 (81.6%), amoxicillin-clavulanic acid 109 (77.3%), cefixime 99 (70.2%), cefepime 93 (66.0%) and tetracycline 91 (64.5%). The extended-spectrum beta-lactamase producing GNB were 69/124 (55.6%). Of which Klebsiella spp. 19 (15.3%) and E. coli 17 (13.7%) were common ESBL producers. Carbapenemase-producing isolates were 8/51(15.7%). Of which Enterobacter, Klebsiella and E. coli were common carbapenemase producers.Conclusion and recommendationMulti-drug resistance and ESBL producing isolates in the present study were high. E. coli and Klebsiella spp. were the most common ESBL producing GNB. Klebsiella spp., Enterobacter spp., E. coli and Citrobacter spp. were typical carbapenemase-producing isolates. Continuous monitoring, antibiotic stewardship and molecular detection of the gene responsible for drug resistance are important means to reduce the spread of drug-resistant pathogens.

Multiple drug resistance bacterial isolates and associated factors among urinary stone patients at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia

BackgroundThe urinary stone and urinary tract infection (UTI) are invariably associated and are frequent causes of morbidity. Date on burden of UTI among urinary stone patients is lacking in Ethiopia. This study was aimed to assess bacterial profile, antimicrobial susceptibility and associated factors among urinary stone patients at the University of Gondar Comprehensive Specialized Hospital.MethodsAn institution based cross sectional study was conducted. Basic sociodemographic data were collected using a structured questionnaire. Bacterial identification of uropathogens and drug susceptibility testing were done following standard microbiological techniques. The data were entered and analyzed using SPSS version-23. Bivariate and multivariate logistic regressions were used to identify possible associated risk factors. Results with P value < 0.05 was considered statistically significant.ResultA total of 300 urinary stone patients were enrolled. Of these, 153 (51%) were male and 261(87%) were urban residents. The overall prevalence of urinary tract infection was 49 (16.3%) (95% CI 12–21%). A high level of resistance was observed to ampicillin, penicillin and trimethoprim-sulfamethoxazole while majority of isolates were most sensitive to nitrofurantoin and ciprofloxacin. Multi-drug resistant isolates were 16/49 (32.7%), 75% of them being Enterobacteriaceae isolates. More than one-third 9/26 (34.6%) of Gram-negative isolates were Extended Spectrum Beta-Lactamase (ESBL) producing E. coli and K. pneumoniae. Being female, history of urinary tract infection and history of drug use were the independent risk factors.ConclusionMost of the bacterial isolates from urinary stone patients were resistant to ampicillin, penicillin and trimethoprim-sulfamethoxazole. E. coli and K. pneumoniae were the most common extended spectrum beta-lactamase producing isolates. Sex, history of urinary tract infection and previous drug use were found to be risk factors. Routine diagnosis of urinary stone patients for urinary tract infection should be promoted and further researches are encouraged.

Investigation on the Genetic Signatures of Antibiotic Resistance in Multi-Drug-Resistant Klebsiella Pneumoniae Isolates From National Guard Hospital, Riyadh.

IntroductionDespite a large number of antibiotics available to treat Klebsiella (K.) pneumoniae (KP), resistance against these antibiotics is ever-increasing and has now become a global threat to human life. The most frequently observed resistant genes in Klebsiella pneumoniae are CTX-M, OXA-48, IMP, and NDM; some are clone-specific while others form a reservoir for infection.MethodsMatrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was employed for the identification of the pathogens and automated VITEK-2 (bioMérieux, Marcy-l'Étoile, France) was used for minimum inhibitory concentration (MIC) determination, followed by polymerase chain reaction (PCR) amplification of target genes and Sanger sequencing of amplicons.ResultsForty-three out of 50 isolates (86%) were OXA gene-positive, and 49 out of 50 (98%) isolates were CTX-M gene positive. Two phenotypes of OXA were identified in 33 samples sequenced, OXA-505 (70%) and OXA-232 (30%). Sixteen isolates (32%) were positive for NDM-1. Twelve isolates were positive for both OXA and NDM. Multilocus sequence typing (MLST) on these isolates showed that they were distributed in 12 sequence types (STs). Thirty-six out of 50 were grouped in four clonal complexes. ST-14 was the predominant genotype.ConclusionThis study has revealed that CTX-M-15 is the most common extended-spectrum beta-lactamase (ESBL) present in almost all isolates. The study also shows the presence of OXA as the main carbapenemase gene, alone or in combination with other carbapenemases such as NDM-1. Multilocus sequence typing revealed the incidence of polyclonal KP pool with ST-14, ST-29, ST-307, and ST-15 being the predominant ones.

Molecular characterization of Danish ESBL/AmpC-producing Klebsiella pneumoniae from bloodstream infections, 2018

ObjectivesThe aim of the study was to molecularly characterize third-generation cephalosporin-resistant Klebsiella pneumoniae isolated from bloodstream infections in Denmark in 2018 using whole-genome sequencing (WGS) data, and to compare these isolates to the most common clones detected in 2006 and 2008. MethodsSixty-two extended-spectrum beta-lactamase (ESBL)/AmpC-producing K. pneumoniae isolates from Danish blood cultures from 2018 were analysed using WGS to obtain multilocus sequence typing (MLST), core genome MLST (cgMLST), resistance profile and phylogeny. These were compared to the most common ESBL K. pneumoniae clones detected in 2006 and 2008. ResultsThe most common ESBL clone was ST15 CTX-M-15, the DHA-1 enzyme was the most common in AmpC isolates, and the OXA-48-like group was the most common carbapenemase. Thirty-nine different sequence types (STs) were found, with the most frequent being ST14, ST15 and ST37, accounting for 24% of the isolates. The isolates were subdivided into 55 complex types (CTs) of which 49 were singletons, with the most frequent being ST14-CT2080. Two of the CTX-M-15-producing isolates from 2018 belonged to the ST15-CT105/CT3078 clone, which was first detected in 2006. ConclusionsThe ESBL/AmpC K. pneumoniae isolates detected in Danish blood cultures belonged to many different types. No dominant clones were circulating in Danish hospitals, but the ST15-CT105/CT3078 CTX-M-15 K. pneumoniae clone was seen 13 years after its first detection.

Antimicrobial Resistance Traits of Escherichia coli Isolated from Dairy Manure and Freshwater Ecosystems Are Similar to One Another but Differ from Associated Clinical Isolates.

Antimicrobial resistance (AMR) is a prevalent global health problem across human and veterinary medicine. The One Health approach to AMR is necessary to mitigate transmission between sources of resistance and decrease the spread of resistant bacteria among humans, animals, and the environment. Our primary goal was to identify associations in resistance traits between Escherichia coli isolated from clinical (n = 103), dairy manure (n = 65), and freshwater ecosystem (n = 64) environments within the same geographic location and timeframe. Clinical E. coli isolates showed the most phenotypic resistance (47.5%), followed by environmental isolates (15.6%) and manure isolates (7.7%), with the most common resistances to ampicillin, ampicillin-sulbactam, and cefotaxime antibiotics. An isolate subset was screened for extended spectrum beta-lactamase (ESBL) production resulting in the identification of 35 ESBL producers. The most common ESBL gene identified was blaTEM-1. Additionally, we found nine different plasmid replicon types including IncFIA-FIB, which were frequently associated with ESBL producer isolates. Molecular phylotyping revealed a significant portion of clinical E. coli were associated with phylotype B2, whereas manure and environmental isolates were more diverse. Manure and environmental isolates were significantly different from clinical isolates based on analyzed traits, suggesting more transmission occurs between these two sources in the sampled environment.

MOLECULAR EPIDEMIOLOGY AND HOSPITAL SPREAD OF CEFTRIAXONE NON‐SUSCEPTIBLE ENTEROBACTERALES IN THE SUNSHINE COAST REGION, AUSTRALIA

Background: Extended spectrum beta‐lactamase (ESBL) and AmpC‐producing Enterobacterales are prevalent worldwide. Poor clinical outcomes, including death, are more common in patients both colonised and infected with these organisms1. Australia is thought to have a comparatively low prevalence of such organisms, however no clear continuous surveillance system exists2. Specific ESBL type has shown significant geographic variation, having different therapeutic and infection control implications for different regions3. Little is known about the prevalence, molecular type and spread of ESBL and AmpC‐producing Enterobacterales among hospitalised patients in the Sunshine Coast region. Aims: To (i) Determine the prevalence and molecular epidemiology of ESBL and AmpC‐producing Enterobacterales, (ii) Develop an in‐house multiplex polymerase chain reaction (PCR) assay to detect common ESBL genes, (iii) Compare our PCR assay to the double disc diffusion method for detection of ESBLs, and to (iv) Establish, with whole genome sequencing (WGS), whether transmission was occurring in our intensive care unit (ICU). Methods: Clinical specimens from adult and paediatric patients culturing Enterobacterales resistant to a third‐generation cephalosporin were collected prospectively from July 2017 to August 2018 at the Sunshine Coast University Hospital laboratory. Antibiotic susceptibility testing was determined using VITEK‐2®. A validated in‐house pentaplex PCR assay was developed and used to detect common beta‐lactamase genes (CTX‐1, CTX‐9, SHV, TEM, CMY). All isolates collected from patients admitted to the ICU were identified and underwent WGS analysis using the Illumina® platform. Results: 295 clinical specimens were identified. Escherichia coli (78.8%), Klebsiella pneumoniae (16.8%) with 1‐2% consisting of Enterobacter spp., Serratia marcescens and Citrobacter freundii. Specimens included urine (53.8%), rectal swab (39.4%), pus (3.0%) and blood (2.4%). Carbapenem resistance was observed in 3.3%. ICU patient specimens made up 18.2%. The majority (87.8%) of isolates had at least one gene identified on PCR testing of which 48.5% produced CTX‐1, 31.2% produced CTX‐9, 15.9% produced SHV, 12.5% produced CMY and 40.6% produced TEM. Approximately forty percent of isolates produced more than one ESBL with the most common being CTX‐1 and TEM coproduction. A small amount (<5%) of isolates were negative on PCR testing. Fourteen (4.7%) isolates that were negative on double disc diffusion had ESBL genes detected by our PCR assay. WGS of non‐identical ICU patient E. coli (n = 44) and K. pneumoniae (n = 10) isolates revealed no strong evidence for transmission of these organisms between patients during this time period, with the smallest single nucleotide polymorphism (SNP) difference being 300 SNPs. Conclusion: Multidrug resistant Gram‐negative organisms are present on the Sunshine Coast and cause significant morbidity. Our real‐time pentaplex PCR assay was able to successfully identify common beta‐lactamase genes, revealing a frequency distribution among our organisms similar to that noted in previous Australian studies (CTX‐1 group most commonly identified). We were not able to identify evidence of ICU transmission, which recognises the success of current infection control practices at our institution.

Phenotypic and Genotypic Characterization of Extended-Spectrum Beta-Lactamases Produced by Escherichia coli Colonizing Pregnant Women.

Introduction Infections caused by extended spectrum beta lactamase (ESBL) producing bacteria continue to be a challenge for choosing the appropriate therapy since they may exhibit coresistance to many other classes of antibiotics. The aim of the study was to screen pregnant women for ESBL producing bacteria in Beirut, Lebanon, to examine their phenotypic and genotypic characterization and to study the association between ESBL colonization with adverse neonatal outcomes. Method In this cross-sectional study, vaginal samples from 308 pregnant women at 35–37 weeks of gestation were studied during a one-year period. The samples were plated on MacConkey agar and selective MacConkey agar supplemented with ceftazidime. Phenotypic confirmation of ESBL production was performed by double-disc synergy test and all isolates were screened by PCR for the resistance genes blaSHV, blaTEM, and blaCTX-M. Clonal relatedness of Escherichia coli isolates was investigated by pulsed-field gel electrophoresis. Results In total, 59 women out of 308 (19.1%) were colonized by ESBL producing gram negative bacteria. Two babies born to mothers colonized with ESBL were diagnosed with sepsis. The susceptibility rates of isolates to other antibiotics were 39% to co-trimoxazole, 49.2% to ciprofloxacin, 91.5% to gentamicin, 18.6% to aztreonam and 35.6% to cefepime. Most of isolates were highly sensitive to meropenem and imipenem, with a susceptibility of 93.2%. PCR was performed on all E. coli isolates to detect the most common ESBL producing genes; blaCTX-M was the predominant gene (90.7%), followed by blaTEM (88.4%) and finally blaSHV (44.2%). PFGE analysis of 34 E. coli isolates revealed 22 distinct clusters showing more than 85% similarity. Conclusion In conclusion, this study showed that Lebanon has a high prevalence of ESBL carriage in pregnant women. Further studies that include a continuous screening of pregnant women and follow up of their newborn clinical status should be conducted to foresee the risk of transmission.

Klinik E.coli İzolatlarında Virulans Faktor Genlerinin ve Geniş Spekturumlu Beta Laktamazların Araştırılması

The aim of this study was to investigate the presence of pap, hly, cnf, sfa, aer and afa virulans genes and extended-spectrum beta lactamase resistance genes in 83 clinical E.coli isolates. In 83 E. coli isolates, sfa, pap, hly, cnf, aer and afa virulence genes and TEM-SHV, CTX-M1 and CTX-M9 wide spectrum beta-lactamase genes were investigated by polymerase chain reaction (PCR) method. Resistance rates of 83 isolates to cefixim, trimethoprim/sulfamethoxazole, piperacillin/tazobactam, amoxicillin/clavulanic acid, ampicillin, ceftriaxone, cefuroxime, cefuroxime axetil, ciprofloxacin, fosfomycin, gentamicin and ceftazidime antibiotics were 65%, 54%, 19%,38, 74%, 75%, 72%, 75%, 68%, 2%, 30%, 49%, respectively. CTX-M1 was the most common extended spectrum beta-lactamase (ESBL) among the ESBL genes investigated in 51 of 83 strains. TEM type beta-lactamase was detected in 33 strains, CTX-M9 and SHV 1 isolates. The most common virulence gene was aer gene and detected in 40 strains, followed by the cnf gene detected in 15 of the isolates. The pap, hly, afa and sfa genes were determined in 15.6%, 12.04%, 9.6% and 15.6% of the isolates, respectively. In addition, 10 different virulence factor gene patterns were observed in 83 E.coli isolates. Virulence gene combinations were observed in 27 strains of aer, 6 strains of cnf-sfa-pap, 1 strains of aer-pap, 2 strains of aer-afa, 6 strains of afa, 3 strain of sfa, 9 strain of aer-cnf-hly, 4 isalates of sfa-pap, 1 strain of hly-pap and 1 strains of pap genes. In conclusion, investigating bacterial pathogenicity associated with UTI will contribute to better medical intervention.

Rapid Detection of Genes Encoding Extended-Spectrum Beta-Lactamase and Carbapenemase in Clinical Escherichia coli Isolates with eazyplex SuperBug CRE System.

Objectives: This study evaluated the diagnostic performance of the eazyplex® SuperBug CRE (eSBCRE) system, based on a loop-mediated isothermal amplification (LAMP), for the detection of the most common extended-spectrum beta-lactamases (ESBL) and carbapenemase genes in 140 clinical isolates of Escherichia coli. Materials and Methods: ESBL (blaCTX-M-1group and blaCTX-M-9group) and carbapenemase (blaKPC, blaVIM, blaNDM, blaOXA-48, and blaOXA-181) genes were detected using the eSBCRE test and compared with the results obtained by PCR, real-time PCR, and phenotypic methods. Results: Concordant results of 100% between PCR/real-time PCR and eSBCRE assays were observed. Two of 140 E. coli isolates were positive for both ESBL and carbapenemase genes according to eSBCRE, PCR, and real-time PCR assays, whereas they were negative in double-disk synergy test. Of 16 E. coli isolates suspected of producing carbapenemase, 9 were positive for 48-oxacillinases (OXA-48) by 30 μg temocillin test, whereas the blaOXA-48 was found only in 1 E. coli isolate by all molecular methods. Maximum threshold time values (minutes:seconds) in the eSBCRE test were 6:00, 11:15, 11:00, and 9:00 for the blaCTX-M-1group, blaCTX-M-9group, blaVIM, and blaOXA-48 genes, respectively. Conclusions: The eSBCRE test based on LAMP method is a reliable, easy-to-use, and timesaving molecular system, which can be successfully used in the routine diagnostic for the rapid detection of the most common ESBL and carbapenemase genes among clinical E. coli isolates.