ESBL Screening Research Articles

Erratum: Bacterial Profile and ESBL Screening of Urinary Tract Infection Among Asymptomatic and Symptomatic Pregnant Women Attending Antenatal Care of Northeastern Ethiopia Region [Corrigendum

[This corrects the article DOI: 10.2147/IDR.S258379.].

Occurrence of the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among fermenting and non-fermenting bacteria from retail goat meat.

The present study was planned to detect the genes encoding carbapenemases, ESBLs and class 1 integron-integrase among bacteria obtained from retail goat meat. Fermenting and non-fermenting bacterial isolates (n=57), recovered from 61 goat meat samples, were identified by 16S rRNA gene sequencing. Antimicrobial susceptibility of isolates was tested by the broth dilution method using ceftazidime, cefotaxime, meropenem and imipenem. Plasmids were isolated and tested for their physical characters. Plasmids were subjected to screening of carbapenemase, ESBL and intI1 gene. Conjugation assay was performed using blaNDM -positive isolates as the donor, and Escherichia coli HB101 as the recipient. Isolates showed the high rates of resistance to ceftazidime (77·2%), cefotaxime (70·2%), meropenem (22·8%) and imipenem (17·5%). They showed variability in number and size (~1 to >20kb) of plasmids. Among all, 1, 4, 13 and 31 isolates showed the blaKPC , blaNDM , blaSHV and blaTEM genes, respectively. The blaKPC-2 gene was observed in one E. coli isolate. The blaNDM-1 gene was detected in Stenotrophomonas maltophilia (n=2), Acinetobacter baumannii (n=1) and Ochrobactrum anthropi (n=1) isolates. These isolates co-harboured the blaTEM and blaSHV genes. The intI1 gene was detected in 22 (38·6%) isolates, and 16 of these isolates showed the carbapenemase and/or ESBL genes. The conjugative movement of blaNDM gene could not be proved after three repetitive mating experiments. The presence of genes encoding carbapenemases and ESBLs in bacteria from goat meat poses public health risks.

Comparison between Exposure of Ciprofloxacin and Cefotaxime on Developing of Escherichia coli ESBL

This study aimed to compare ciprofloxacin and cefotaxime exposure to develop ESBL producing Escherichia coli (E. coli). A total of 16 isolates of cefotaxime sensitive E. coli and ciprofloxacin were exposed to ciprofloxacin and cefotaxime for 14 days using the Kirby-Bauer antibiotic disc diffusion method. Colonies that grew on the edge of the inhibiting zone were exposed each day by the same method. Furthermore, we observed the occurrence of resistance to cefotaxime as ESBL screening test. Isolates were resistant, the following day the ESBL was confirmed by the Modified Double Disk Sinergy Test (MDDST) method using Cefotaxime (CTX), Ceftazidime (CAZ), Aztreonam (ATM), and Amoxilin Clavulanate (AMC) antibiotic discs. From 16 isolates of ESBL producing E. coli exposed to ciprofloxacin, it was obtained 4 (25%) to ESBL E. coli. ESBL production occurred after E. coli was exposed to ciprofloxacin on days 5, 6, 7, and 12. While those exposed to cefotaxime none becomes ESBL E. coli. There was no difference between ciprofloxacin and cefotaxime exposure to develop ESBL producing E. coli (p=0.101; Chi-square).

Bacterial Profile and ESBL Screening of Urinary Tract Infection Among Asymptomatic and Symptomatic Pregnant Women Attending Antenatal Care of Northeastern Ethiopia Region.

BackgroundUrinary tract infection is a common cause of morbidity in pregnant women. Emergence of antimicrobial resistance particularly ESBL production among bacterial uropathogens is increasing and becoming principal cause of treatment failure. The aim of this study was to determine the bacterial profile, their antimicrobial susceptibility patterns, risk factors and identify ESBL-producing bacterial uropathogens.Patients and MethodsA hospital-based cross-sectional study was conducted in the Northeastern Ethiopia region. A total of 323 pregnant women were included and structured questionnaire was used to collect sociodemographic and risk factor-related data. About 10mL freshly voided midstream urine specimen was collected, transported and processed according to standard operating procedures. The data obtained were entered into SPSS version 22 and descriptive statistics, chi-square, bivariate and multivariate logistic regression analyses were performed. P-value ≤0.05 with corresponding 95% confidence interval were considered for statistical significance.ResultsThe overall prevalence of UTI was 15.5% (50/323). The predominant bacterial isolates were Escherichia coli 17 (33.3%) followed by coagulase-negative staphylococci 15 (30.0%) and Staphylococcus aureus 14 (27.5%). Previous history of UTI (AOR=8.824, 95% CI: 3.769,20.654, P<0.001) and history of catheterization (AOR=3.270, 95% CI: 1.316,8.122, P=0.011) were significantly associated with the occurrence of bacterial UTI. Gram-negative isolates showed high level of resistance to ampicillin 12 (60.0%) and relatively low level of resistance to nitrofurantoin 5(25.0%), norfloxacin 5 (25.0%) and ceftazidime 3 (15.0%). Gram-positive uropathogens showed higher resistance for penicillin 29 (93.5%) and trimethoprim-sulfamethoxazole 23 (79.3%) whereas all isolates were sensitive 29 (100.0%) to nitrofurantoin. Moreover, multidrug resistance was observed among 41 (80.4%) of the isolates, and 3 (15.8%) of isolated gram-negative bacteria were ESBL producers.ConclusionHigh prevalence of bacterial UTI and MDR for commonly prescribed drugs were observed with significant number of ESBL producers. Therefore, instant UTI culture assessment of pregnant women, especially those having possible risk factors such as previous histories of UTI and catheterization; moreover, appropriate prescription and use of antibiotics are necessary.

Screening of OXA-244 producers, a difficult-to-detect and emerging OXA-48 variant?

OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France. Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST. Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%-22.5%), 54% (95% CI = 43.3%-63.4%) and 99% (95% CI = 93.8%-100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%-78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8). Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%-78% of them using ESBL-specific screening media.

Performance of the Check-Direct ESBL Screen for BD MAXTM for detection of asymptomatic faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae

ObjectivesAccurte diagnostic methods are crucial for the detection of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E). Besides culture-based gold-standard methods, new molecular gene detection tests are reaching the market. The aim of this study was to investigate the performance of the direct quantitative PCR (qPCR)-based methods Check-Direct ESBL and CPE Screen for BD MAXTM in relation to traditional culture-based methods for detection of ESBL-E faecal carriage. MethodsFaecal samples were collected from healthy adult volunteers. Samples were cultured on chromogenic ESBL agar plates and were screened for ESBL-producing Escherichia coli and Klebsiella pneumoniae. Confirmed ESBL- and AmpC-producing isolates were further analysed using whole-genome sequencing. In addition, faecal samples were analysed using Check-Direct ESBL and CPE Screen for BD MAXTM and the results were compared with the gold-standard culture-based method. ResultsOf 176 faecal samples examined, 11 (6.3%) grew ESBL-producing E. coli or K. pneumoniae isolates. Among 173 analysed samples, Check-Direct ESBL Screen for BD MAXTM detected 22 (12.7%) ESBL-positive samples. No carbapenemase-producing isolates were detected. Two culture-positive samples remained negative with Check-Direct ESBL Screen for BD MAXTM. Culture-negative but qPCR-positive discrepancy was observed in 12 samples (6.9%). Altogether, concordant results were obtained for 158 samples (91.3%; 9 positive and 149 negative). ConclusionCheck-Direct ESBL Screen for BD MAXTM is a fast screening method for ESBL carriage. However, several discrepant results were observed, which hinders interpretation. More clinical samples should be tested in combination with culture to evaluate the true benefits of this method.

Drug-resistant organisms are common in fecal surveillance cultures, predict bacteremia and correlate with poorer outcomes in patients undergoing allogeneic stem cell transplants.

With the increasing incidence of multidrug-resistant (MDR) organisms and high mortality rates associated with these infections, we describe the spectrum of the major drug-resistant pathogens identified in fecal surveillance, and re-visit the use of fecal surveillance in predicting infection with these organisms post-allogeneic stem cell transplant. Data from allogeneic stem cell transplant recipients with common drug-resistant strains of bacteria in fecal surveillance (Escherichia coli, Klebsiella spp., and Enterococcus spp.) were compared with recipients who did not have the same in fecal surveillance cultures. Baseline characteristics and post-transplant outcomes including similar drug resistance in blood cultures, severe sepsis, and 100-day transplant-related mortality were compared. Multivariate analysis using logistic regression model was used to determine independent predictors of outcome. In 232 transplants, the prevalence of common drug-resistant isolates in fecal surveillance cultures was 57.7% (134 out of 232 patients-with a single isolate in 115 and ≥2 isolates in the remaining 19 patients. A total of 164 drug-resistant isolates were obtained from 134 patients. Of the 164 isolates, 133 (81%) were positive for ESBL screening, 19 (11.5%) for carbapenem-resistant organisms (CRO) screening, 12 (7.3%) for VRE screening. Patients who had common drug-resistant pathogens detected in fecal surveillance have significantly higher subsequent blood culture positivity with drug resistance, as well as higher 100-day mortality. Factors influencing 100-day mortality included patient's age (P=.001), drug resistance positivity in blood (P<.001), drug resistance in fecal surveillance (P=.011), use of an alternate donor (other than fully matched sibling) (P<.001), GVHD grade 3-4 (P<.001), and severe sepsis (P<.001). On multivariate analysis, only use of an alternate donor (0.024), severe sepsis (P<.001), and grade 3-4 GVHD (P<.001) retained significance in predicting 100-day mortality. Organisms resistant to 3rd generation cephalosporins are frequently seen on fecal surveillance in the pre-transplant setting and are associated with a higher incidence of drug-resistant organisms in subsequent blood cultures (not limited to the same drug resistance pattern as seen in fecal surveillance). Drug-resistant organisms in fecal surveillance are associated with poorer outcomes following allogeneic stem cell transplant and may be used as a guide to identify patients at risk of subsequently developing a drug-resistant organism in blood.

Determination of ESBL Production &amp; Antibiotic Resistance Profile of Escherichia Coli Isolated from Patients with Urinary Tract Infections: A Study from Tertiary Care Hospital

Urinary tract infection (UTI), is defined as a disease caused by invasion of urinary tract by microorganisms. Majority of UTI cases are due to bacterial infection constitute about 95% of total UTI cases. About 80% of UTI cases are caused by E.coli producing extended spectrum ?-lactamase (ESBL) producing isolates. In recent years limitations in treating infections caused by multidrug resistant organisms has increased. This study aims to determine ESBL production of E. coli cases from a tertiary care hospital. Methodology: A total 358 midstream urine samples were collected by random sampling method during March 2015 to June 2018. Identification, antibiotic sensitivity testing, performed according to standard protocol following Clinical and Laboratory Standard Institute (CLSI) guidelines, 2013. Screening for ESBL producing E.coli isolates performed using ceftazidime further confirmation done by phenotypic disc diffusion test using combined disc method using ceftazidime (30µg) &amp; ceftazidime/ clavulanic acid (30/10 µg) as per CLSI guidelines. Results: Total 358 specimens processed for urine culture. Gram negative bacilli isolated from 123(34.35 %), out of which 68 (55.28%) were E.coli, 19 (15.44%) K. pneumoniae, 15 (12.19%), Pseudomonas spp. 08 (6.50%), Citrobacter spp and Acinetobacter spp, 03 (2.43%), Proteus mirabilis, 01 (0.81%) Proteus vulgaris and Enterobacter respectively. Out of 68 isolates of E.coli, 65 (95.58%) were MDR, ESBL was detected in 31 (47.69%) out of these 65 isolates. Out of these 31 cases 19 (61.29%) were female and 12 (38.70%) were male cases. Conclusion: This study concludes 47.69% ESBL producing MDR E. coli were isolated from UTI cases with female predominance.

Phenotypic and Molecular Characterization of β-Lactamases among Enterobacterial Uropathogens in Southeastern Nigeria.

Little is known about the molecular basis of antibiotic resistance among uropathogens in Southeast Nigeria. The aim of the study was to characterize enterobacterial uropathogens with respect to drug resistance. One hundred (100) enterobacterial uropathogens were studied. Their antibiotic susceptibility patterns were evaluated using disk diffusion, screened, and confirmed phenotypically for the presence of β-lactamases: ESBL, AmpC, carbapenemase, and MBLs. Screen positives were further tested for various β-lactamase genes by PCR. Our isolates showed variable resistance to most drugs tested. Out of the 58 ESBL screen positive E. coli, 35 were confirmed positive with PCR. The predominant ESBL gene was blaTEM while blaSPM was the most prevalent among MBL genes. Forty-six percentage of the screen positive Salmonella isolates coharbored blaTEM + SHV genes. Nine of the 10 ESBL screen positive K. pneumoniae were phenotypically and PCR positive. Three isolates of K. pneumoniae were positive for MBL genes. All the 10 C. freundii were positive for ESBL genes. The study showed high prevalence of drug-resistant genes among the enterobacterial uropathogens. Majority of the uropathogens harbored >1 antibiotic-resistant gene, and the most predominant gene was ESBL (blaTEM) followed by the MBL (SPM) gene.

Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella Species in Pediatric Patients Visiting International Friendship Children's Hospital, Kathmandu, Nepal.

Introduction:Emergence and spread of antimicrobial resistance (AMR) is a global threat and significantly affects the treatment options for common infectious diseases. Inappropriate use of antibiotics, particularly third-generation cephalosporins, has contributed to the development of AMR. This study aims to determine the prevalence of extended-spectrum β-lactamase (ESBL) production in Escherichia coli and Klebsiella species isolated from various clinical samples.Methods:This cross-sectional study was conducted at International Friendship Children’s Hospital, Kathmandu, Nepal, from August 2017 to January 2018. A total of 1443 samples that included urine, pus, wound swab, endotracheal tip, catheter tip, and blood were collected from pediatric patients below 15 years and processed by standard microbiological methods. Following sufficient incubation, isolates were identified by colony morphology, gram staining, and necessary biochemical tests. Identified bacterial isolates were then tested for antibiotic susceptibility test by modified Kirby-Bauer disk diffusion method and were subjected to ESBL screening by using 30 µg cefotaxime and ceftazidime. The ESBL production was confirmed by combination disk method.Results:From a total of 103 nonduplicated clinical isolates, E. coli (n = 79), Klebsiella pneumoniae (n = 18), and Klebsiella oxytoca (n = 6) were isolated from different clinical specimens. Of which, 64 (62.1%) exhibited multidrug resistance, and 29 (28.2%) were ESBL producers. All ESBL-producing isolates were resistant toward ampicillin, cefotaxime, ceftriaxone, and ceftazidime. Most ESBL producers were susceptible toward imipenem (89.7%; 26/29), nitrofurantoin (82.8%; 24/29), piperacillin/tazobactam (79.3%; 23/29), and amikacin (72.4%; 21/29).Conclusions:A high prevalence of multidrug-resistant ESBL organisms was found in this study among pediatric patients. Treatment based on their routine identification and susceptibility to specific antibiotics is critical to halt the spread of AMR and ESBL.

Coexistence of β-Lactamases in Community-Acquired Infections in a Tertiary Care Hospital in India.

Introduction The trends of β-lactamases producing Enterobacteriaceae is ever increasing, and limited studies have reported investigating coexistence of β lactamases in Enterobacteriaceae. A cross-sectional study after approval from the Institutional Ethical committee was conducted between June 2014 and May 2016 in community-acquired infections due to multidrug-resistant organisms in our tertiary care. Nonrepetitive clinical samples from the out-patient department (OPD) were processed for bacteriological culture and identification of Enterobacteriaceae. An antibiotic susceptibility test, screening, and phenotypic confirmation for ESBLs and carbapenemases and AmpC producers were performed to check for coexistence of these enzymes. Results Nonrepetitive clinical specimens processed for culture and identification in our hospital revealed 417 positive isolates in community acquired infections which were multidrug-resistant organisms, and on screening for β-lactamases, 293 isolates were positive for one of the three beta lactamases, ESBL, AmpC, or carbapnemases. Coproduction of ESBL and MBL was seen in 5 isolates, 35 isolates showed coproduction of ESBL and AmpC enzymes, and AmpC and MBL coproduction was exhibited in only in 5 isolates. Conclusions Coexistence of ESBLs, AmpC producers, and carbapenemases has been described. Continuous monitoring and surveillance and proper infection control and prevention practices will limit the further spread of these superbugs within the hospital and beyond.

893. The SHIELD Orange County Project: A Decolonization Strategy in 35 Hospitals and Nursing Homes Reduces Multi-Drug-Resistant Organism (MDRO) Prevalence in a Southern California Region

BackgroundPatient movement between hospitals, nursing homes (NH), and long-term acute care facilities (LTACs) contributes to MDRO spread. SHIELD OC is a regional decolonization collaborative among adult facilities with high patient sharing designed to reduce countywide MDRO prevalence. We report pre- and post-intervention MDRO colonization prevalence.MethodsDecolonization included chlorhexidine bath (CHG) (4% liquid or 2% cloth) and twice-daily nasal swab 10% povidone–iodine (PI). LTAC and NH used CHG for all baths and PI 5 days on admission and Monday–Friday every other week. Patients in contact precautions (CP) at hospitals had daily CHG and 5-days PI on admission. Point-prevalence screening for MRSA, VRE, ESBL, and CRE using nares, axilla/groin, and peri-rectal swabs was conducted pre-intervention (September 2016–March 2017) and post-intervention (August 2018–April 2019); 50 random LTAC and 50 CP hospitalized patients were sampled; for NH up to 50 were sampled at baseline and all residents post-intervention. Raw impact of the intervention was assessed by the average change in colonization prevalence, with each facility carrying equal weight. Generalized linear mixed models (GLM) stratified by facility type were used to assess the impact on MDRO colonization when clustering by facility.ResultsAcross 35 facilities (16 hospitals, 16 NHs, 3 LTACs), the overall MDRO prevalence was reduced 22% in NHs (OR 0.58, P < 0.001), 34% LTACs (OR = 0.27, P < 0.001), and 11% CP patients (OR = 0.67, P < 0.001, Table 1). For MRSA, raw reductions were 31% NHs (OR = 0.58, P < 0.001), 39% LTACs (OR = 0.51, P = 0.01), and 3% CP patients (OR = 0.88, P = NS). For VRE, raw reductions were 40% NHs (OR = 0.62, P = 0.001), 55% LTACs (OR = 0.26, P < 0.001), and 15% CP patients (OR = 0.67, P = 0.004). For ESBLs, raw reductions were 24% NHs (OR = 0.65, P < 0.001), 34% LTACs (OR = 0.53, P = 0.01), and 26% CP patients (OR = 0.64, P < 0.001). For CRE, raw reductions were 24% NHs (OR = 0.70, P = NS), and 23% LTACs (OR = 0.75, P = NS). CRE increased by 26% in CP averaged across hospitals, although patient -level CRE declined 2.4% to 1.8% (OR = 0.74, P = NS).ConclusionMDRO carriage was common in highly inter-connected NHs, LTACs and hospitals. A regional collaborative of universal decolonization in long-term care and targeted decolonization of CP patients in hospitals led to sizeable reductions in MDRO carriage. Disclosures All Authors: No reported Disclosures.

2142. Comparison of Molecular-Based vs. Conventional Culture-Based Screening Methods for Detection of Carriers of Extended-Spectrum β-Lactamases (ESBL) and Carbapenemases-Producing Enterobacteriacae (CPE)

BackgroundActive surveillance and contact precautions may prevent cross-transmission of ESBL-producing Enterobacteriaceae and CPE. Culture-based methods delaying results might increase risk of cross-transmission, and lead to unnecessary preemptive contact precautions. This observational cohort study compared rapid Loop-Mediated Isothermal Amplification (LAMP) assays to conventional culture-based methods for ESBL and CPE screening of critically ill patients.MethodsThis study was conducted in the adult ICUs at Geneva University Hospitals. We collected consecutive rectal ESwabs routinely performed, either for admission screening of high-risk patients or once weekly routine screening of all patients hospitalized in the ICU. Eazyplex® SuperBug CRE system (Amplex Biosystems) assays were performed directly on rectal Eswabs according to the manufacturer’s instructions. For the conventional culture-based method, we used chromID® ESBL agar (ESBL) coupled with chromID®OXA-48. Discordant specimens were retested using disk diffusion tests and the same LAMP assay on isolates. Microbiological turn-around times (TAT; from the reception in the laboratory to result notification) were collected.ResultsOverall, 290 rectal ESwabs were analyzed. ESBL and CPE prevalence were 16.7% and 1.0%, respectively. Three discordant isolates could not be further investigated and considered as LAMP false positive. Adjusted analytical performances were for CPE: 100% (95IC 100–100%) sensitivity, 99.6% (99.0–100%) specificity, 75% (32.6–100%) PPV, and 100% (100–100%) NPV, and for ESBL: 85% (73.9–96.1%) sensitivity, 98.8% (97.4–100%) specificity, 91.9% (83.1–100%) PPV, and 97.6% (95.7–99.5%) NPV. A decrease in TAT was observed when comparing LAMP screening assay against conventional method (50.3 hours vs. 6.2 hours; Figure 1). Figure 2 shows time reductions comparing both screening strategies.ConclusionScreening strategies based on LAMP could fasten discontinuation of unnecessary pre-emptive isolation time for patients at risk and earlier implementation of contact precautions for previously unknown carriers of ESBL or CPE. Disclosures All authors: No reported disclosures.

Distribution of fosfomycin and AmpC β-lactamase resistance genes in urinary Escherichia coli isolates obtained from patients admitted to an educational hospital in Ahvaz, southwest Iran

BackgroundEscherichia coli is the most important etiologic agent of urinary tract infections (UTIs). Today, the emergence of antibiotic resistance in UTI-causing E. coli isolates is increasing. This study aimed to investigate the extended-spectrum β-lactamases (ESBLs) production, AmpC enzymes, and fosfomycin resistance genes in E. coli clinical isolates obtained from patients with UTIs. MethodsFrom December 2018 to June 2019, the E. coli isolates were collected from patients with UTI who were referred to Golestan teaching hospital in Ahvaz, Iran. Then, the antimicrobial susceptibility testing, ESBL and AmpC β-lactamase production were performed by disk diffusion method. Finally, the polymerase chain reaction was done to investigate the presence of fosfomycin resistance (fosC2, fosA3) and AmpC (blaCMY-2, blaACC) genes. ResultsIn total, 90 E. coli isolates were collected. The most effective antibiotics were fosfomycin, nitrofurantoin and imipenem with 86 (95.5%), 82 (91.1%), and 78 (86.7%) susceptibility, respectively. AmpC beta-lactamase was confirmed in 18 (20%) isolates. Moreover, 63 (90%) isolates were found as ESBL producers and 6 (6.6%) had both AmpC and ESBL β-lactamases. The frequency rates of blaCMY-2 and blaACC resistance genes in AmpC-producing isolates was 12 (13.3%) and 15 (83.3%), respectively. The fosC2 and fosA3 genes were not detected in E. coli isolates. ConclusionCurrent study revealed the emergence of E. coli isolates harboring ESBL and AmpC enzymes in southwest region of Iran. This study highlighted the need of our laboratories to adopt an appropriate method for continuous screening of ESBL and AmpC in clinical isolates on a daily basis.

Mutant Prevention Concentration of Ciprofloxacin against Klebsiella pneumoniae Clinical Isolates: An Ideal Prognosticator in Treating Multidrug-Resistant Strains.

Background Fluoroquinolone-resistant Klebsiella pneumoniae poses a therapeutic challenge when implicated in urinary tract infections, pyelonephritis, pneumonia, skin infections, osteomyelitis, and respiratory infections. The mutant prevention concentration (MPC) represents a concentration threshold above which increase of resistant mutants occurs rarely. The aim of the present study is to determine the MPC among ciprofloxacin-resistant K. pneumoniae clinical isolates. Materials and Methods A total of 240 clinical isolates of K. pneumoniae were collected from a tertiary care hospital. The MPCs were determined for 24 selected strains using an inoculum of 1010 CFU/ml in Müller–Hinton agar plates with serial/various concentrations (0.003–100 μg) of ciprofloxacin. In addition to the MPC, phenotypic screening for ESBL, AmpC, and carbapenemase was performed. The detection of qnr genes for 24 isolates and DNA sequencing for six isolates were performed. Results Ciprofloxacin resistance was observed in 19.6% of the K. pneumoniae clinical isolates. Among the ciprofloxacin-resistant isolates, 14 isolates showed an MPC value of more than 100 μg. The MPC ranged between 100 μg and 20 μg for ciprofloxacin-resistant isolates. ESBL producers and qnr gene-producing strains had a high MPC. 11 isolates showed the presence of either qnrB or qnrS genes. None of the samples showed the presence of the qnrA gene. Conclusion From our study, we infer that ESBL producers and qnr gene-possessing strains are frequently resistant to ciprofloxacin. Estimation of the MPC in the case of multidrug-resistant isolates in the clinical setup may help in treating these drug-resistant strains.

Characterisation of Extended β-Lactamases and Plasmid Mediated Quinolones Resistancein Escherichia Coli from Shelter Dogs

The aim of this study was to determine the prevalence of β-lactamase (TEM, SHV, OXA), extended-spectrum β-lactamase (ESBL) and genes encoding plasmid mediated resistance to quinolones (PMQR) in extended spectrum cephalosporin (ESC)-resistant Escherichia coli isolated from dog faeces from two shelters in the North-East of Romania. Eighty-eight faecal samples from healthy dogs were analysed by cultivation on Brilliance ESBL medium (Oxoid, UK), followed by phenotipic ESBL screening using combination disc test (CDT). Identification of the E. coli strains was performed by uidA/uspA gene PCR. Susceptibility testing was performed on Mueller-Hinton Agar, with β-lactam and non-β-lactam agents. Identification of β-lactamase genes (blaCTX-M, blaTEM, blaSHV, blaOXA) and PMQR genes (qnrA, qnrB and qnrS) was performed by PCR as previously described. Twenty eight ESC-resistant E. coli (31.81%) were obtained and (n=21/28, 75%) of these were confirmed as ESBLs and showed resistance to cefpodoxime (n=21/28, 75%), amoxicillin/clavulanic acid (n=19/21; 90.48%), and enrofloxacin (n=8/21; 38.09%). Predominant ESBL types were CTX-M-1 (n=15/17, 88.24%) and CTX-M-9 (n=2/17, 11.76%) enzymes. TEM and SHV enzymes were identified in 17.86% and 14.29% of the ESC-resistant isolates, whilst some isolates (n=4) carried only blaTEM and blaSHV. The prevalence of PMQR genes was 28.57% of the 28 ESC resistant isolates, consisting of qnrS (62.5%) and qnrB (37.5%). These findings indicate a high prevalence of ESBLs and PMQR associated resistance E. coli in the normal faecal microbiota of dogs from shelters, which carries the risk for dissemination of these resistance genes to other animals, human or the environment.

Molecular Typing of Klebsiella pneumoniae Isolated from Medical Centers in Kermanshah Using Pulse Field Gel Electrophoresis

Background: Klebsiella pneumoniae is one of the important opportunistic pathogens causing serious infections, both community and hospital-acquired, with high resistance to antibiotics. Objectives: We aimed to determine the molecular typing of K. pneumoniae isolates using pulse field gel electrophoresis (PFGE). Methods: Sixty-two consecutive non-duplicate K. pneumoniae isolates were isolated from clinical specimens in three hospitals of Kermanshah, west of Iran. A total of K. pneumoniae was confirmed by API-20E kit. Antibiotic susceptibility testing and ESBL screening were performed following the genotyping of isolates using PFGE. The pattern of DNA bands was examined by the 6.6 version of the Gelcompar II (Applied Maths, Belgium). The calculation of similarities was done by the Dice coefficient, and the unweighted paired group method was used for cluster analysis (UPGMA). Results: Fifty-eight percent of isolates were phenotypically ESBL positive. Twenty-nine pulsotypes (X1-29) were determined with various genotype patterns. Four clones (including 18 isolates) showed a close genetic relationship and were from ICU, burn and infectious wards in one hospital. Pulsotypes X15 to X29, each presented a unique genotype and the majority of these strains (> 50%) were from the emergency ward. Four isolates were non-typable. Conclusions: Our results indicate the genotypic variation of K. pneumoniae isolates, which may suggest the different origins for isolates, as well as ongoing changes of bacterial genome. However, the presence of the isolates with similar genotype may show a common origin and consequently, the dissemination of strains in hospital wards, especially in ICU and burn wards. Therefore, Kermanshah hospitals need to establish an effective infectious control system to manage and monitor the spread of resistant strains.

High prevalence of CTX-M-1 group in ESBL-producing enterobacteriaceae infection in intensive care units in southern Chile

Enterobacteria-producing extended-spectrum β-lactamases (ESBL) play an important role in healthcare infections, increasing hospitalization time, morbidity and mortality rates. Among several ESBLs that emerge from these pathogens, CTX-M-type enzymes had the most successful global spread in different epidemiological settings. Latin America presents high prevalence of CTX-M-2 in ESBL-producing enterobacterial infections with local emergence of the CTX-M-1 group. However, this high prevalence of the CTX-M-1 group has not yet been reported in Chile. The aim of this study was to identify ESBLs among enterobacteria isolated from clinical samples of critically ill patients from southern Chile. One-hundred thirty seven ESBL-producing bacteria were isolated from outpatients from all critical patient units from Hernán Henríquez Aravena Hospital. Phenotype characterization was performed by antibiogram, screening of ESBL, and determination of minimum inhibitory concentration (MIC). PCR was used for genetic confirmation of resistance. Molecular typing was performed by ERIC-PCR. ESBL-producing isolates were identified as Klebsiella pneumoniae (n=115), Escherichia coli (n=18), Proteus mirabilis (n=3), and Enterobacter cloacae (n=1), presenting multidrug resistance profiles. PCR amplification showed that the strains were positive for blaSHV (n=111/81%), blaCTX-M-1 (n=116/84.7%), blaTEM (n=100/73%), blaCTX-M-2 (n=28/20.4%), blaCTX-M-9 (0.7%), blaPER-1 (0.7%), and blaGES-10 (0.7%). The multiple production of ESBL was observed in 93% of isolates, suggesting high genetic mobility independent of the clonal relationship. The high frequency of the CTX-M-1 group and a high rate of ESBL co-production are changing the epidemiology of the ESBL profile in Chilean intensive care units. This epidemiology is a constant and increasing challenge, not only in Chile, but worldwide.

A novel disk-based detection method with superior sensitivity for β-lactamase production in third-generation cephalosporin-resistant Enterobacteriaceae

ObjectiveCurrent phenotypic methods for extended-spectrum β-lactamase (ESBL), AmpC β-lactamase (AmpC), and carbapenemases fail to detect isolates that co-produce other classes of β-lactamases. In this study, we have developed a novel assay (Applied Multiplex Disk Method: AMU-DM) for the phenotypic detection and identification of β-lactamases produced by Enterobacteriaceae. MethodsWe evaluated the performance of the method by comparison with PCR results for 78 Enterobacteriaceae clinical isolates that were positive by the ESBL screening test and negative by the ESBL confirmation test. Additionally, one NCTC strain and four ATCC strains were also included in the test population for the study as reference. ResultsFor 79/83 (95%) isolates tested, the AMU-DM results matched those obtained by PCR. The concordance rates were 31/31 (100%), 11/11 (100%), 3/3 (100%), 0/1 (0%), 15/15 (100%), 16/19 (84%), and 3/3 (100%) for AmpC, ESBL and AmpC co-production, Klebsiella pneumoniae carbapenemase (KPC), KPC and ESBL co-production, metallo β-lactamase (MBL), MBL and ESBL co-production, and MBL and AmpC co-production, respectively. ConclusionThe AMU-DM is convenient to perform, economical, and highly sensitive in identifying ESBLs, AmpCs, and carbapenemases. Our method may be useful in clinical settings for the implementation of relevant infection control measures and for surveillance purposes.

Rapid Detection of Extended-Spectrum β-Lactamases (ESBL) and AmpC β-Lactamases in Enterobacterales: Development of a Screening Panel Using the MALDI-TOF MS-Based Direct-on-Target Microdroplet Growth Assay.

Introduction: Antibiotic resistant bacteria are a growing concern worldwide. Extended-spectrum β-lactamases (ESBL) represent the most common resistance mechanism of Gram-negative bacteria against β-lactams, underlining the need for adequate diagnostic methods that provide reliable information in the shortest time possible. AmpC, a less prevalent but increasingly relevant class of β-lactamases, pose an additional challenge as their detection is complex. Here, we present an ESBL and AmpC screening panel employing the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA).Materials and Methods: Four reference strains recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used to develop the panel, which was further validated on 50 clinical Enterobacterales isolates resistant to third generation cephalosporins. The panel relies on the synergistic effect between ESBL and/or AmpC β-lactamase inhibitors and cephalosporins, which indicates β-lactamase production. Microdroplets containing the tested microorganism, cephalosporins in different concentrations and inhibitors were pipetted onto an MBT Biotarget and incubated for 3 or 4 h at 35 ± 1°C. Afterward, the liquid medium was removed and the material adhered to the spots was analyzed by MALDI-TOF MS. Synergy was detected by determining and comparing the minimum inhibitory concentrations of the tested cephalosporins with and without β-lactamase inhibitors. Data were interpreted following a diagnostic algorithm proposed by EUCAST in order to establish a final diagnosis. In comparison, PCR, broth microdilution (BMD) and combination disk tests (CDT) were performed.Results: Compared to the PCR results, the following positive and negative percent agreement values (PPA/NPA) were obtained for each resistance mechanism: ESBL, 94.44/100%; AmpC, 94.44/93.75% and ESBL+AmpC, 100/100%. These results, obtained after 4 h of incubation, were comparable with those of BMD and showed a higher accuracy than CDT.Discussion: We propose a novel phenotypic method for detection of ESBL and AmpC β-lactamases in Enterobacterales that provides reliable results in a short time, representing a promising alternative to the diagnostic techniques currently available. This easy-to-perform approach has potential for being implemented in routine laboratories, contributing to the further diversification of mass spectrometry technology into other fields such as antibiotic resistance testing.