ESBL-producing Enterobacteriaceae Isolates Research Articles

Primary Carrier Status and Alteration in the Colonization of the Extended-Spectrum β-Lactamase Producing Enterobacteriaceae Among Children in Pediatric Intensive Care Unit During the Hospital Stay

Background: Widespread and inappropriate use of antibiotics has led to an increase in antibiotic-resistant bacteria worldwide. Extended-spectrum β-lactamases (ESBLs) are among the most important resistance mechanisms in members of Enterobacteriaceae, which can pose a threat to patients. Objectives: This study aimed to investigate the carrier status and alteration in the fecal transmission of ESBL-producing Enterobacteriaceae (ESBL-E) on admission and during the hospital stay, as well as its correlation with the usage of antibiotics among children in a pediatric intensive care unit (PICU). Molecular typing between the primary and secondary isolates was done to detect the homotypic clonal strains. Methods: Demographic and medical data of PICU children were collected, and the carrier status of ESBL-E was investigated in pairs of their rectal swab samples at the admission and discharge time. Detection of ESBL phenotype and antimicrobial susceptibility to 12 antibiotics were performed by double-disk synergy and disc diffusion methods, respectively. Polymerase chain reaction for detection of blaTEM, blaSHVblaPER, blaCTX-M, and blaVEB genes was performed using specific primers. The phylogenetic relations were analyzed by the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) method. Results: Extended-spectrum β-lactamase-producing Enterobacteriaceae was detected in 48% of the samples at admission and 42% at discharge time. The highest frequency of resistance was observed in cephazolin, amoxicillin-clavulanic acid, and ampicillin. Children with a history of meropenem administration showed a significantly higher frequency of meropenem resistance Enterobacteriaceae in their samples. Moreover, the administration of metronidazole increased the isolation of ESBL-Escherichia species in hospitalized children. The most common gene associated with the ESBL phenotype was blaCTX-M, while blaPER and blaVEB were not detected in the ESBL-E isolates. The phylogenetic analysis did not confirm the occurrence of the cross-transmission of ESBL-E in the patients during hospitalization. Conclusions: Our results showed a high frequency of ESBL-E in the feces of children upon admission to the PICU, which did not change significantly during the hospital stay. Although the prescription of metronidazole showed an association with the enrichment of ESBL-E due to observed diversity in the molecular types and resistance phenotypes of the isolates, the community seems to be the primary source of ESBL-E transmission in children. Further investigations are needed to understand the role of hospital stay in the colonization and enrichment of ESBL-E in the intestinal tract of children and their association with infections during the medical interventions in the PICU.

Cefotaxime/sulbactam plus gentamicin as a potential carbapenem- and amikacin-sparing first-line combination for neonatal sepsis in high ESBL prevalence settings.

Infection with ESBL-producing Enterobacteriaceae infection is ubiquitous in some neonatal ICUs and increasing levels of antibiotic resistance are a cause for urgent concern. Delineation of bacterial and viral sepsis can be challenging, often leading to patients receiving empirical antibiotics without or whilst waiting for a definitive causal diagnosis. Empirical therapy is often dependent on broad-spectrum 'Watch' antibiotics, contributing to further resistance. ESBL-producing Enterobacteriaceae clinical isolates found to have caused neonatal sepsis and meningitis underwent a detailed in vitro screening including susceptibility testing, chequerboard combination analysis and hollow-fibre infection model dynamic analyses using combinations of cefotaxime, ampicillin and gentamicin in combination with β-lactamase inhibitors. Additivity or synergy was found for all antibiotic combinations against seven Escherichia coli and three Klebsiella pneumoniae clinical isolates. Cefotaxime or ampicillin plus sulbactam combined with gentamicin was able to consistently inhibit the growth of ESBL-producing isolates at typical neonatal doses, and the combination cleared the hollow-fibre infection model system of organisms resistant to each agent alone. The combination of cefotaxime/sulbactam and gentamicin was consistently bactericidal at clinically achievable concentrations (Cmax of 180, 60 and 20 mg/L for cefotaxime, sulbactam and gentamicin, respectively). The addition of sulbactam to cefotaxime or ampicillin to the typical first-line empirical therapy could obviate the need for carbapenems and amikacin in settings with high ESBL-infection prevalence.

Phenotypic characterization and antibiotic suceptibility patterns of extended spectrum beta-lactamase producing enterobacteriaceae

The rise of Enterobacteriaceae strains that produce an extended spectrum b-lactamase (ESBL) has become a global concern for epidemiological surveillance and the prevention of nosocomial acquired infections in the modern era. The choice of appropriate antibiotics to be employed in the treatment of infections brought on by ESBL-producing bacteria relies greatly on the detection and identification of these ESBLs in the laboratory. Limitations in ESBL detection have aided in the unbridled emergence of bacterial resistance and constitute a major health concern. Isolation and identification of ESBL among Enterobacteriaceae by phenotypic methodswith their Antibiogram.Phenotypic techniques were used to identify ESBL-producing Enterobacteriaceae (ESBLs-E) isolates from diverse clinical samples. Kirby Baur disc diffusion technique was performed to determine antimicrobial's susceptibility.Among 212 Enterobacteriaceae isolates 124(58.3%) were positive for ESBL production. E.coli(74.5%) & K.pneumoniae (52.2%) two main isolates that produce ESBLs. Maximum ESBL producing Enterobacteriaceae isolates were obtained from blood samples 82% (41/50) followed by urine 59 % (62/105). Meropenem (96.7%), Amikacin (82.1%), and Cefoxitin were most susceptible antibiotics for ESBL-producing isolates while high resistance was observed in ceftazidime (62%), followed by Ciprofloxacin (60%). The majority of ESBLs-E was mostly found in urine and blood samples.It was observed that E.coli produced the most ESBLs. There was a high prevalence of ESBLs-E in tertiary care hospital of central India. Therefore, strong infection control strategies must be implemented in hospital settings

Phenotypic detection of ESBL-producing Enterobacteriaceae using combined disk diffusion, ESBL HiCrome agar, and E-test: A comparative study

Background: Extended-spectrum beta-lactamases (ESBLs) are in a rising trend in recent years, creating confusion for physicians to choose appropriate antimicrobials for treatment.Aim: The aim of the study is to detect ESBL-producing Enterobacteriaceae by using rapid detection tests such as combined disk diffusion, ESBL E-test strips (based on cefotaxime and cefotaxime+clavulanate), and ESBL HiCrome agar and compare the efficacy of these tests.Materials and Methods: Samples were processed using conventional methods. Bacterial antibiotic susceptibility testing was done on Mueller-Hinton agar according to the Clinical and Laboratory Standards Institute guidelines. All Enterobacteriaceae isolates were subjected to ESBL HiCrome agar, combined disk diffusion, and E-test.Results: Out of 5299 samples, 2097 (39.57%) were culture-positive, and 200 (9.5%) Enterobacteriaceae isolates were obtained. The majority of the isolates were Escherichia coli (67.5%), followed by Klebsiella pneumoniae (25%), Proteus mirabilis (3.5%), Proteus vulgaris (2%), and Citrobacter freundii (2%). 29.5% of all Enterobacteriaceae isolates were found to be ESBL producers by combined disk diffusion, ESBL HiCrome agar, and E-test methods, which showed 100% concordance.Conclusion: It is important to identify ESBL-producing Enterobacteriaceae from clinical samples for the judicious use of antibiotics. For early detection of ESBL-producing Enterobacteriaceae isolates, combined disk diffusion, ESBL HiCrome agar, and E tests were found to be equally effective in detecting ESBL production.

Genetic Determinants of Resistance among ESBL-Producing Enterobacteriaceae in Community and Hospital Settings in East, Central, and Southern Africa: A Systematic Review and Meta-Analysis of Prevalence.

Background The world prevalence of community and hospital-acquired extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing tremendously. Bacteria harboring ESBLs are currently the number one critical pathogens posing a major threat to human health. Objective To provide a summary of molecular evidence on the prevalence of ESBL-producing Enterobacteriaceae (ESBL-E) and associated genes at community and hospital settings in East, Central, and Southern African countries. Methods We conducted a systematic literature search on PubMed and Google Scholar databases for the available molecular studies on ESBL-E in hospitals and community settings in East, Central, and Sothern Africa (ECSA). Published studies in English language involving gene characterization of ESBLs from human samples in hospital and community settings were included in the review, inception to November 2019. A random effect meta-analysis was performed to estimate the prevalence of ESBL-E. Results A total of 27 studies involving molecular characterization of resistance genes from 20,225 ESBL-E isolates were included in the analysis. Seventy-four percent of all studies were hospital based, 15% were based in community settings, and others were done in both hospital and community settings. Of all the studies, 63% reported E. coli as the dominant isolate among ESBL-E recovered from clinical samples and Klebsiella pneumoniae was reported dominant isolates in 33% of all studies. A random pooled prevalence of ESBL-E was 38% (95% CI = 24–53%), highest in Congo, 92% (95% CI = 90–94%), and lowest in Zimbabwe, 14% (95% CI = 9–20%). Prevalence was higher in hospital settings 41% (95% CI = 23–58%) compared to community settings 34% (95% CI = 8–60%). ESBL genes detected from clinical isolates with ESBL-E phenotypes in ECSA were those of Ambler molecular class A [1] that belongs to both functional groups 2be and 2d of Bush and Jacob classification of 2010 [2]. Majority of studies (n = 22, 81.5%) reported predominance of blaCTX-M gene among isolates, particularly CTX-M-15. Predictors of ESBL-E included increased age, hospital admissions, previous use of antibiotics, and paramedical use of herbs. Conclusion Few studies have been conducted at a molecular level to understand the genetic basis of increased resistance among members of ESBL-E in ECSA. Limited molecular studies in the ECSA region leave a gap in estimating the burden and risk posed by the carriage of ESBL genes in these countries. We found a high prevalence of ESBL-E most carrying CTX-M enzyme in ECSA with a greater variation between countries. This could be an important call for combined (regional or global) efforts to combat the problem of antimicrobial resistance (AMR) in the region. Antibiotic use and hospital admission increased the carriage of ESBL-E, while poor people contributed little to the increase of AMR due to lack of access and failure to meet the cost of healthcare compared to high income individuals.

Dietary Risk of bla ESBL Producing Multidrug Resistant Enterobacteriaceae and their Inhibition by Artemisia herba-alba and Thymus algeriensis Essential Oils

The main objective of this study is to highlight the dietary risk of the prevalence of bla ESBL producing multidrug-resistant Enterobacteriaceae in raw milk, besides evaluating the effect of Artemesia herba-alba and Thymus algeriensis essential oils against bla ESBL producing Enterobacteriaceae isolates from raw milk. Isolation and molecular identification of ESBL producing Enterobacteriaceae and bla TEM, bla SHV, bla CTX-M genes encoding resistance were investigated by PCR and sequencing. The chemical composition of oils was effectuated by GC/MS. Antibacterial activity was tested by disc diffusion and microdilution methods. Six ceftazidime-resistant Enterobacteriaceae were identified; four of them were confirmed as ESBL producers and were found to be multi-drug resistant. bla SHV and bla TEM were found to be having the most dominant gene encoding resistance. The results of GC/MS analysis bestowed 31 and 35 compounds for A. herba-alba and T. algeriensis respectively. Klebsiella pneumonia SB6 (17.04 mm for A. herba-alba and 16.22 mm for T. algeriensis) revealed the highest zones of inhibition. MIC values were 1.56-12.5 mg/mL for both essential oils, while the MBC values were 6.25-25 mg/mL (A. herba-alba) and 12.5-25 mg/mL (T. algeriensis).

Detection and characterization of ESBL-producing Enterobacteriaceae from the gut of subsistence farmers, their livestock, and the\xa0surrounding environment in rural Nepal

The increasing trend of gut colonization by extended-spectrum β-lactamase (ESBL) producing Enterobacterales has been observed in conventional farm animals and their owners. Still, such colonization among domesticated organically fed livestock has not been well studied. This study aimed to determine the gut colonization rate of ESBL-producing Enterobacteriaceae and carbapenemase-producing Enterobacteriaceae (CPE) among rural subsistence farming communities of the Kaski district in Nepal. Rectal swabs collected by systematic random sampling from 128 households of subsistence farming communities were screened for ESBL-producing Enterobacteriaceae and CPE by phenotypic and molecular methods. A total of 357 (57%) ESBL-producing Enterobacteriaceae isolates were obtained from 626 specimens, which included 97 ESBL-producing Enterobacteriaceae (75.8%) from 128 adult humans, 101 (79.5%) from 127 of their children, 51 (47.7%) from 107 cattle, 26 (51%) from 51 goats, 30 (34.9%) from 86 poultry and 52 (42%) from 127 environmental samples. No CPE was isolated from any of the samples. blaCTX-M-15 was the most predominant gene found in animal (86.8%) and human (80.5%) isolates. Out of 308 Escherichia coli isolates, 16 human and two poultry isolates were positive for ST131 and were of clade C. Among non-cephalosporin antibiotics, the resistance rates were observed slightly higher in tetracycline and ciprofloxacin among all study subjects. This is the first one-health study in Nepal, demonstrating the high rate of CTX-M-15 type ESBL-producing Enterobacteriaceae among gut flora of subsistence-based farming communities. Gut colonization by E. coli ST131 clade C among healthy farmers and poultry birds is a consequential public health concern.

Molecular detection of plasmid-derived AmpC β-lactamase among clinical strains of Enterobacteriaceae in Bahrain.

BACKGROUND:Enterobacteriaceae with AmpC β-lactamase are multidrug-resistant organisms and represent a significant challenge to patient care. This study aims to determine the prevalence of plasmid-derived AmpC β-lactamase among extended spectrum β-lactamases (ESBL)-producing Enterobacteriaceae strains in Bahrain.METHODS:It was a cross-sectional study. A total of 185 ESBL-producing Enterobacteriaceae isolates were recovered from clinically significant specimens from January 2018 to December 2019. The samples underwent initial screen for cefoxitin resistance by disc diffusion test and subsequent phenotypic confirmation of AmpC production with phenyl boronic acid assays as well as genotypic analysis by multiplex polymerase chain reactions for AmpC subtypes. Drug-resistant features of these clinical isolates were also examined.RESULTS:Twenty-nine ESBL-producing Enterobacteriaceae isolates were cefoxitin resistant. Phenotypic and genotypic analyses confirmed that 8 and 12 cefoxitin-resistant isolates are AmpC positive, respectively. These AmpC producers are multidrug resistant, and Escherichia coli is the dominant strain among them.CONCLUSIONS:Plasmid-mediated spread of AmpC is present in clinically relevant Enterobacteriaceae species in Bahrain. Rational antimicrobial therapy against these multidrug-resistant organisms and continued surveillance of antimicrobial resistance mechanisms among the clinical isolates are recommended for optimal patient care.

In vitro activity of flomoxef on ESBL-producing Enterobacteriaceae isolates from surgical site infections – A Multicentre Study in Malaysia

In vitro activity of flomoxef on ESBL-producing Enterobacteriaceae isolates from surgical site infections – A Multicentre Study in Malaysia

Molecular Detection of Extended-Spectrum β-Lactamases- Producer Serratia marcescens Causing Neonatal Sepsis in Iraq

Serratia marcescens is an important nosocomial pathogen that causes a variety of infections, especially urinary tract and bloodstream infections. The emergence and spread of multidrug-resistant Serratia marcescens producing extended-spectrum beta-lactamases is a threat to public health worldwide at present. Extended-spectrum β-lactamases (ESβLs) including TEM, SHV, and CTX-M are the predominant types that confer resistance to beta-lactam group of antibiotics. Many reports have been investigated ESBL-producing isolates of Enterobacteriaceae in Iraq. However, there are few studies concerned about ESβL-producing Serratia marcescens particularly, detection of ESBLs encoding genes. Therefore this study aimed to identify ESβLs encoding genes in Serratia marcescens isolates from a neonatal intensive care unit. Fifty isolates were identified phenotypically using the VITEK® 2 compact system. For confirming the identification of bacterial strains, molecular detection of housekeeping LuxS gene was done using species-specific designed primers. Antibiogram was performed using the VITEK® 2 compact system. A phenotypic confirmatory test for ESβLs producers was performed using a combination disc method. The ESβLs encoding genes, including blaTEM, blaSHV, and blaCTX-M, were amplified using a PCR-based technique; the amplified products of some selected isolates were sequenced. Molecular detection of isolates using PCR-based amplification of the LuxS gene showed that all isolates possessed this gene. The patterns of antimicrobial resistance for isolates under study showed very high resistance to cephalosporins, while they were susceptible to carbapenem drugs and tigecycline. Findings based on the PCR technique showed that the prevalence of ESβLs encoding genes of isolates was 13 (26%), 31 (62%), and 46 (92%) for blaTEM, blaSHV, and blaCTX-M respectively. In the present study, it was concluded that blaCTX-M gene was the most prevalent ESβLs-encoding gene among ESβLs producing Serratia marcescens isolates.

Genetic characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from wastewater and river water in Tunisia: predominance of CTX-M-15 and high genetic diversity.

Aquatic environments are crucial hotspots for the dissemination of antibiotic resistant microorganisms and resistance genes. Thus, the purpose of this study was to investigate the occurrence and the genetic characterization of cefotaxime-resistant (CTXR) Enterobacteriaceae at a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river (Rouriche River, downstream from WWPP) located in Tunis City, during 2017-2018. We collected 105 and 15 water samples from the WWPP and the Rouriche River, respectively. Samples were screened to recover ESBL-producing Enterobacteriaceae (ESBL-E) and isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, plasmid types and molecular typing (multilocus sequence typing, MLST). Among 120 water samples, 33 and 4 contained ESBL-producing E. coli and K. pneumoniae isolates, respectively. Most isolates were multidrug resistant and produced CTX-M-15 (28 isolates), CTX-M-1 (4 isolates), CTX-M-55 (2 isolates), CTX-M-27 (one isolate), SHV-12 (one isolate) and VEB beta-lactamases (one isolate). All K. pneumoniae were CTX-M-15-positive. Four colistin-resistant isolates were found (MIC 4-8μg/ml), but they were negative for the mcr genes tested. Class 1 integrons were detected in 21/25 trimethoprim/sulfamethoxazole-resistant isolates, and nine of them carried the gene cassette arrays: aadA2 + dfrA12 (n = 4), aadA1 + dfrA15 (n = 2), aadA5 + dfrA17 (n= 2) and aadA1/2 (n = 1). The IncP and IncFIB plasmids were found in 30 and 16 isolates, respectively. Genetic lineages detected were as follows: E. coli (ST48-ST10 Cplx, ST2499, ST906, ST2973 and ST2142); K. pneumoniae: (ST1540 and ST661). Our findings show a high rate of CTX-M-15 and high genetic diversity of ESBL-E isolates from WWPP and receivingriver water.

Prevalence and associated factors for carriage of Enterobacteriaceae producing ESBLs or carbapenemase and methicillin-resistant Staphylococcus aureus in Hong Kong community

ObjectivesWe conducted a cross-sectional study in Hong Kong community to estimate the carriage prevalence, associated factors and genotypes of extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E), methicillin-resistant Staphylococcus aureus (MRSA) and carbapenemase-producing Enterobacteriaceae (CPE). MethodsSeemingly healthy subjects were asked to provide nasal, handprint and stool samples from March to April 2017. Isolates were characterized by molecular methods. We used multivariable logistic regression models within a generalized estimating equation framework to identify risk factors for ESBL-E carriage. Characteristics of MRSA/CPE carriage were summarized. ResultsThe prevalence of ESBL-E, MRSA and CPE were 52.8% (104/197), 2.5% (5/197) and 0.5% (1/197) respectively. Most ESBL-E isolates were E. coli (85.6%; 113/132). Most ESBL genes belonged to blaCTX−M-G9 (68.9%) and blaTEM (53.0%) types. Self-reported antibiotic consumption (≥2 courses) in the past six months was associated with ESBL-E carriage (adjusted odds ratio: 4.71–5.47). ConclusionsAbundance of ESBL-E in the community are causes of concern, and antibiotic use is associated with its carriage. Presence of MRSA and CPE in community members without clear healthcare exposure hints on a change in their epidemiology. This study establishes a baseline to formulate infection control policies and future studies in combating antimicrobial resistance.

Prevalence of the carbapenem-heteroresistant phenotype among ESBL-producing Escherichia coli and Klebsiella pneumoniae clinical isolates.

Carbapenem-heteroresistant (cHR) Enterobacteriaceae strains have been reported worldwide; however, the prevalence among clinical ESBL-producing Enterobacteriaceae isolates obtained from patients with repeated hospital admissions remains largely unknown. Heteroresistance was screened by disc diffusion and confirmed by a modified population analysis profiling (PAP) method against ertapenem, imipenem, meropenem and ceftolozane/tazobactam. MIC testing was performed by broth microdilution against carbapenems and a panel of agents with potential activity against ESBL-producing strains. One hundred and seventy-three ESBL-producing meropenem-susceptible Escherichia coli and Klebsiella pneumoniae isolates were selected for testing. A total of 519 bacteria/carbapenem combinations were screened by disc diffusion; 84 combinations were identified as cHR. Modified PAP confirmed 70 bacteria/carbapenem combinations as heteroresistant; most (63%, 44/70) confirmed cHR colonies grew within the ertapenem zone of inhibition, followed by imipenem (30%, 21/70), then meropenem (7%, 5/70). In total, one-third of the unique patient isolates (32%, 55/173) were identified as being heteroresistant to at least one carbapenem; of those patients, 16% (9/55) had a carbapenem-non-susceptible isolate on subsequent visits. Only two cHR isolates screened positive for ceftolozane/tazobactam heteroresistance (1%, 2/173), of which one was confirmed heteroresistant by modified PAP. cHR isolates were more likely to be collected from a non-urinary source (e.g. respiratory) compared with non-cHR isolates (31% versus 19%, P = 0.02). MIC distributions of all tested antibiotic agents did not differ between non-cHR and cHR isolates. Our findings raise concerns for the continued use of carbapenems as first-line therapy for ESBL infections and for the potential selection for strains with full carbapenem resistance.

Decreasing prevalence of contamination with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in retail chicken meat in the Netherlands.

Retail chicken meat is a potential source of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). In the past decade, vast national efforts were undertaken to decrease the antibiotic use in the veterinary sector, resulting in a 58% decrease in antibiotic sales in the sector between 2009 and 2014. This decrease in antibiotic use was followed by a decrease in ESBL-E prevalence in broilers. The current study investigates the prevalence of contamination with ESBL-E in retail chicken meat purchased in the Netherlands between December 2013 and August 2015. It looks at associations between the prevalence of contamination with ESBL-E and sample characteristics such as method of farming (free-range or conventional), supermarket chain of purchase and year of purchase. In the current study, 352 chicken meat samples were investigated for the presence of ESBL-E using selective culture methods. Six samples were excluded due to missing isolates or problems obtaining a good quality sequence leaving 346 samples for further analyses. Of these 346 samples, 188 (54.3%) were positive for ESBL-E, yielding 216 ESBL-E isolates (Escherichia coli (n = 204), Klebsiella pneumoniae (n = 11) and Escherichia fergusonii (n = 1)). All ESBL-E isolates were analysed using whole-genome sequencing. The prevalence of contamination with ESBL-E in retail chicken meat decreased from 68.3% in 2014 to 44.6% in 2015, absolute risk difference 23.7% (95% confidence interval (CI): 12.6% - 34.1%). The ESBL-E prevalence was lower in free-range chicken meat (36.4%) compared with conventional chicken meat (61.5%), absolute risk difference 25.2% (95% CI: 12.9% - 36.5%). The prevalence of contamination with ESBL-E varied between supermarket chains, the highest prevalence of contamination was found in supermarket chain 4 (76.5%) and the lowest in supermarket chain 1 (37.8%). Pairwise isolate comparisons using whole-genome multilocus sequence typing (wgMLST) showed that clustering of isolates occurs more frequently within supermarket chains than between supermarket chains. In conclusion, the prevalence of contamination with ESBL-E in retail chicken in the Netherlands decreased over time; nevertheless, it remains substantial and as such a potential source for ESBL-E in humans.

A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying blaSHV-12 from horses in Sweden.

The aim of this study was to investigate the occurrence of the newly described transferable colistin resistance gene mcr-9 in extended-spectrum β-lactamase (ESBL)-producing clinical Enterobacteriaceae isolates from horses in Sweden. A total of 56 whole-genome sequenced ESBL-producing Enterobacteriaceae isolates from horses were subjected to in silico detection of antimicrobial resistance genes and identification of plasmid replicons types. The colistin minimum inhibitory concentration (MIC) for mcr-positive isolates was determined by broth microdilution. Relatedness between Enterobacteriaceae carrying mcr genes was determined by multilocus sequence typing (MLST) and core genome MLST. Thirty ESBL-producing Enterobacteriaceae isolates from horses were positive for the colistin resistance gene mcr-9. These isolates included Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca and Citrobacter freundii and belonged to diverse MLST sequence types within each species. Two of the mcr-9-containing isolates originated from the same horse. All mcr-9-positive isolates had colistin MICs below or equal to the EUCAST epidemiological cut-off value of 2 mg/L and were negative for the two potential regulatory genes qseB-like and qseC-like for mcr-9. Except for one isolate carrying only blaTEM-1B, all of the isolates carried blaSHV-12 and blaTEM-1B, and were all considered multidrug-resistant as they harboured genes encoding resistance to aminoglycosides, chloramphenicol, fosfomycin, macrolides, quinolones, sulfonamides, trimethoprim and tetracyclines. Plasmid replicon types IncHI2 and IncHI2A were detected in all mcr-9-positive isolates. The occurrence of mcr-9 was common among clinical ESBL-producing Enterobacteriaceae isolates from horses in Sweden and was linked to the ESBL-encoding gene blaSHV-12 and plasmid replicon types IncHI2 and IncHI2A.

Real world incidence estimation methodologies used for surveillance of HIV in repeat blood donors

This study was performed to characterize CTX-M type extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 μg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of blaCTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred blaCTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.

Emerging clinical role of pivmecillinam in the treatment of urinary tract infections caused by Extended Spectrum βeta-lactamase (ESBL) producing Enterobacteriaceae.

Extended Spectrum βeta-lactamase (ESBL)-producing Enterobacteriaceae causing urinary tract infections (UTIs) appear resistant to many common oral agents. There is a growing need to discover new antibiotics to combat with emerging antibiotic resistance problem. Until the discovery of new antimicrobials, we can bring back forgotten antibiotics to our clinical formulary. Pivmecillinam (prodrug of mecillinam), an oral antimicrobial agent is effective against ESBL producing organisms. We analysed the sensitivity rates of ESBL-producing Enterobacteriaceae from urine samples to mecillinam and to document if pivmecillinam is a suitable alternative option in the treatment of UTI. This retrospective study was conducted from September 2015 to September 2017. Data were collected from the pathology information system. Antimicrobial sensitivity testing on ESBL-producing Enterobacteriaceae isolates was carried out by disc diffusion method in accordance with The European Committee on Antimicrobial Susceptibility Testing. A total of 986 ESBL-producing Enterobacteriaceae were tested for mecillinam during the study period. Of 986 organisms, Escherichia coli was the most common organism (889); followed by Klebsiella species (71) and others Enterobacteriaceae (26). Mecillinam sensitivity was found in 96% Escherichia coli (855/889 isolates), 83% Klebsiella species (59/71 isolates) and 88% other Enterobacteriaceae (23/26 isolates). Overall 95% (935/986 isolates) of ESBL-producing urinary isolates were sensitive to mecillinam. Pivmecillinam appears to be suitable option to treat ESBL-producing Enterobacteriaceae causing uncomplicated UTI. Our results showed low resistance rate to mecillinam. We recommend the use of pivmecillinam in uncomplicated UTIs because of ESBL-producing Enterobacteriaceae. More studies on in vitro activity of mecillinam against ESBL producing organism and its use and clinical outcome should be tried in future.

Prevalence of the crpP gene conferring decreased ciprofloxacin susceptibility in enterobacterial clinical isolates from Mexican hospitals.

This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

Effects of a specific nutrient combination on ESBL resistance.

Background and aimExtended-spectrum beta-lactamases are the main cause of resistance in Enterobacteriaceae to beta lactam antibiotics. The aim of this study was to evaluate the antimicrobial effect of EpiQuercican supplement, combined with different antimicrobial agents, on ESBL-producing isolates and determine the underlying molecular mechanism of resistance in these isolates.Materials and methodsEleven ESBL producing Enterobacteriaceae isolates were collected from Saudi Arabia hospitals between 2016 and 2017 and disk diffusion test was performed in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines to determine the susceptibility of the isolates to 5 different antibiotics in the presence of EpiQuercican supplement. Polymerase chain reaction was performed for detection of ESBL genes, and efflux pump inhibitor was used to study the mechanism of resistance in these isolates.ResultsThe best synergistic effect was obtained when the supplement was combined with carbapenems followed by 4th generation cephalosporins. Either no effect or antagonistic effect was seen with most of the isolates when the supplement was added to the 3rd generation of cephalosporins. Among the tested genes responsible for ESBL production in this study, our results indicated the predominance of TEM genes (73%) followed by CTX-M genes (9%). As for the mechanism of resistance in ESBL isolates, 4 isolates showed to use efflux pumps as their main mechanism of resistance.ConclusionThe EpiQuercican supplement showed some promising results, yet its antibacterial mechanism of action needs to be elucidated further.

Characterization of CTX-M type ESBL-producing Enterobacteriaceae isolated from asymptomatic healthy individuals who live in a community of the Okinawa prefecture, Japan.

This study was performed to characterize CTX-M type extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2μg/ml of cefotaxime and incubated at 37°C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of blaCTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E.coli B2 were E.coli B2-ST131. Three CTX-M type ESBL-producing E.coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E.coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred blaCTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E.coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.