AmpC-producing Isolates Research Articles

PHENOTYPIC DETECTION OF ESBL AND AMPC BETA LACTAMASES AMONG GRAM-NEGATIVE ISOLATES FROM CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL

Objectives: Drug-resistant Gram-negative bacilli expressing extended-spectrum beta-lactamases (ESBLs) and AmpC pose a serious therapeutic threat in nosocomial infections. Cost-effective screening methods are a boon to patients. This study aims to detect gram-negative bacilli and their antibiotic sensitivity patterns, as well as detect the ESBL and AmpC-producing isolates among Gram-negative bacilli. Methods: A prospective study was conducted with 150 samples. Gram-negative bacilli were isolated, and their antibiotic sensitivity tests were performed by the Kirby–Bauer disk diffusion method. Potential ESBL producers were screened using Ceftazidime disc, and AmpC producers were screened by Cefoxitin discs by the disc diffusion method. ESBL producers were confirmed by the combined disc diffusion assay method using ceftazidime and ceftazidime/Clavulanic acid disc. AmpC producers were confirmed by the Cefoxitin Cloxacillin Double Disc Synergy Test. Results: About 38% of 150 samples were gram-negative bacilli, of which 40.35% were Escherichia coli, followed by Pseudomonas aeruginosa (35.08%). Maximum sensitivity by E. coli was found toward imipenem, meropenem, and cotrimoxazole. P. aeruginosa showed maximum sensitivity toward piperacillin/tazobactam, imipenem, meropenem, and ceftazidime. 28.07% of Gram-negative isolates were ESBL producers, with E. coli (11 isolates) being the maximum, and 15.78% were AmpC producers, with E. coli (four isolates) being the maximum. Seven isolates were both ESBL and AmpC producers. Conclusion: Routine screening and timely reporting of ESBL and AmpC producers help in preventing the spread of multidrug-resistant strains. Antibiotic resistance surveillance helps in the implementation of strict infection control and prevention practices.

Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum β-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

he occurrence and characteristics of Extended Spectrum- and AmpC-β-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

Detection of plasmid-mediated AmpC β-lactamases in Klebsiella pneumoniae clinical isolates from Bushehr province, Iran.

Plasmid-mediated AmpC producers are considered an increasing concern. The aim of this study was to investigate the prevalence of plasmid-mediated AmpC β-lactamases (pAmpCs) in Klebsiella pneumoniae isolates. A total of 228 clinical isolates of K. pneumoniae were collected in Bushehr province, Iran, from December 2017 to February 2019. Cefoxitin disks were applied for screening AmpC-producing isolates. Furthermore, 3 phenotypic confirmatory tests including combine disk test (CDT), double disk synergy test (DDST) and modified three dimensional test (M3DT) were used. Finally, the presence of pAmpC genes was tested by multiplex PCR. We identified 18 pAmpC-KP isolates among the 228 isolates (7.9%): 12 DHA (66.6%) and 6 CMY (33.3%). In the present study only 47% of cefoxitin-resistant isolates were pAmpC producers. The sensitivity of CDT, DDST, and M3DT was 89%, 67% and 100% and the specificity was 90%, 90% and 85%, respectively. In addition, M3DT displayed a higher rate of efficiency (92%) than CDT (89%) and DDST (79%) in detecting plasmid-meditated AmpC producers. DHA was the most prevalent pAmpC beta-lactamase in this study. DDST and CDT tests proved inefficient to detect two and six pAmpC producers, respectively, while M3DT represented the best overall performance.

Characterization of Carbapenem-Resistant Escherichia Coli and Klebsiella : A Role For Ampc-Producing Isolates

Aim: This study aimed to investigate the role of AmpC enzymes in carbapenem resistance among AmpC/extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Escherichia coli and Klebsiella spp. Methods: Fifty-six bacterial strains that were AmpC producers were examined. The antibiotic susceptibility test was performed by the disk diffusion and E-test. The prevalence of the plasmid carbapenemase was determined using PCR. Results: The resistance to meropenem in the AmpC+/ESBL+ group was 64%, higher than that reported for the AmpC-/ESBL+ group. Ten isolates of the carbapenem-resistant AmpC producers were negative for carbapenemase-encoding genes. Conclusion: Carbapenem resistance among AmpC-producing isolates with negative results for carbapenemase-encoding genes potentially demonstrates the role of AmpC enzymes among these isolates.

In Vitro Activity of Ceftibuten-Avibactam against β-Lactamase-Positive Enterobacterales from the ATLAS Global Surveillance Program.

Ceftibuten is an established, oral, third-generation cephalosporin in early clinical development in combination with an oral prodrug of avibactam for the treatment of complicated urinary tract infections, including acute pyelonephritis. We evaluated the in vitro activity of ceftibuten-avibactam against 1,165 Enterobacterales isolates selected from the 2016-2020 ATLAS global surveillance program based upon their β-lactamase genotype, β-lactam-susceptible phenotype, species identification, and specimen source (95.8% urine). MICs were determined by CLSI broth microdilution. Avibactam was tested at a fixed concentration of 4 μg/mL. Molecular methods were used to identify β-lactamase genes. Ceftibuten-avibactam inhibited 90% (MIC90) of ESBL-producing (n = 645), KPC-producing (n = 60), chromosomal AmpC-positive (n = 100), OXA-48-like-producing (n = 50), and acquired AmpC-producing (n = 110) isolates at concentrations of 0.12, 0.5, 1, 2, and 4 μg/mL, respectively. At concentrations of ≤1 and ≤8 μg/mL, ceftibuten-avibactam inhibited 98.4 and 99.2% of ESBL-positive isolates; 96.7 and 100% of KPC-positive isolates; 91.0 and 99.0% of chromosomal AmpC-positive isolates; 86.0 and 96.0% of OXA-48-like-positive isolates; and 85.5 and 91.8% of acquired AmpC-positive isolates. Against ESBL-producing, KPC-producing, chromosomal AmpC-positive, OXA-48-like-producing, and acquired AmpC-producing isolates, ceftibuten-avibactam was 256-, 128-, >64-, >32-, and > 16-fold more potent than ceftibuten alone. The potency of ceftibuten-avibactam was 4-fold greater than ceftazidime-avibactam against ESBL-producing (ceftibuten-avibactam MIC90, 0.12 μg/mL; ceftazidime-avibactam MIC90, 0.5 μg/mL) and KPC-producing (0.5 μg/mL; 2 μg/mL) isolates, equivalent to ceftazidime-avibactam (MIC90, 2 μg/mL) against OXA-48-like-producing isolates, 2-fold less active than ceftazidime-avibactam (1 μg/mL; 0.5 μg/mL) against chromosomal AmpC-positive isolates, and 4-fold less active than ceftazidime-avibactam (4 μg/mL; 1 μg/mL) against acquired AmpC-producing isolates. Continued development of ceftibuten-avibactam appears justified.

Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC.

Beta-lactamase (BL) production is a major public health problem. Although not the most frequent AmpC type, AmpC-BL is increasingly isolated, especially plasmid AmpC-BL (pAmpC-BL). The objective of this study was to review information published to date on pAmpC-BL in Escherichia coli and Klebsiella pneumoniae, and on the epidemiology and detection methods used by clinical microbiology laboratories, by performing a systematic review using the MEDLINE PubMed database. The predictive capacity of a screening method to detect AmpC-BL using disks with cloxacillin (CLX) was also evaluated by studying 102 Enterobacteriaceae clinical isolates grown in CHROMID ESBL medium with the addition of cefepime (FEP), cefoxitin (FOX), ertapenem (ETP), CLX, and oxacillin with CLX. The review, which included 149 publications, suggests that certain risk factors (prolonged hospitalization and previous use of cephalosporins) are associated with infections by pAmpC-BL-producing microorganisms. The worldwide prevalence has increased over the past 10 years, with a positivity rate ranging between 0.1 and 40%, although AmpC was only detected when sought in a targeted manner. CMY-2 type has been the most prevalent pAmpC-BL-producing microorganism. The most frequently used phenotypic method has been the double-disk synergy test (using CLX disks or phenyl-boronic acid and cefotaxime [CTX] and ceftazidime) and the disk method combined with these inhibitors. In regard to screening methods, a 1-µg oxacillin disk with CLX showed 88.9% sensitivity, 100% specificity, 100% positive predictive value (PPV), 98.9% negative predictive value (NPV), and 98.9% validity index (VI). This predictive capacity is reduced with the addition of extended-spectrum beta-lactamases, showing 62.5% sensitivity, 100% specificity, 100% PPV, 93.5% NPV, and 94.1% VI. In conclusion, there has been a worldwide increase in the number of isolates with pAmpC-BL, especially in Asia, with CMY-2 being the most frequently detected pAmpC-BL-producing type of microorganism. Reduction in its spread requires routine screening with a combination of phenotypic methods (with AmpC inhibitors) and genotypic methods (multiplex PCR). In conclusion, the proposed screening technique is an easy-to-apply and inexpensive test for the detection of AmpC-producing isolates in the routine screening of multidrug-resistant microorganisms.

Genetic but No Phenotypic Associations between Biocide Tolerance and Antibiotic Resistance in Escherichia coli from German Broiler Fattening Farms.

Biocides are frequently applied as disinfectants in animal husbandry to prevent the transmission of drug-resistant bacteria and to control zoonotic diseases. Concerns have been raised, that their use may contribute to the selection and persistence of antimicrobial-resistant bacteria. Especially, extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli have become a global health threat. In our study, 29 ESBL-/AmpC-producing and 64 NON-ESBL-/AmpC-producing E. coli isolates from three German broiler fattening farms collected in 2016 following regular cleaning and disinfection were phylogenetically characterized by whole genome sequencing, analyzed for phylogenetic distribution of virulence-associated genes, and screened for determinants of and associations between biocide tolerance and antibiotic resistance. Of the 30 known and two unknown sequence types detected, ST117 and ST297 were the most common genotypes. These STs are recognized worldwide as pandemic lineages causing disease in humans and poultry. Virulence determinants associated with extraintestinal pathogenic E. coli showed variable phylogenetic distribution patterns. Isolates with reduced biocide susceptibility were rarely found on the tested farms. Nine isolates displayed elevated MICs and/or MBCs of formaldehyde, chlorocresol, peroxyacetic acid, or benzalkonium chloride. Antibiotic resistance to ampicillin, trimethoprim, and sulfamethoxazole was most prevalent. The majority of ESBL-/AmpC-producing isolates carried blaCTX-M (55%) or blaCMY-2 (24%) genes. Phenotypic biocide tolerance and antibiotic resistance were not interlinked. However, biocide and metal resistance determinants were found on mobile genetic elements together with antibiotic resistance genes raising concerns that biocides used in the food industry may lead to selection pressure for strains carrying acquired resistance determinants to different antimicrobials.

Wildlife as Sentinels of Antimicrobial Resistance in Germany?

The presence of bacteria carrying antimicrobial resistance (AMR) genes in wildlife is an indicator that resistant bacteria of human or livestock origin are widespread in the environment. In addition, it could represent an additional challenge for human health, since wild animals could act as efficient AMR reservoirs and epidemiological links between human, livestock and natural environments. The aim of this study was to investigate the occurrence and the antibiotic resistance patterns of several bacterial species in certain wild animals in Germany, including wild boars (Sus scrofa), roe deer (Capreolus capreolus) and wild ducks (family Anatidae, subfamily Anatinae) and geese (family Anatidae, subfamily Anserinae). In the framework of the German National Zoonoses Monitoring Program, samples from hunted wild boars, roe deer and wild ducks and geese were collected nationwide in 2016, 2017, and 2019, respectively. Fecal samples were tested for the presence of Salmonella spp. (in wild boars and wild ducks and geese), Campylobacter spp. (in roe deer and wild ducks and geese), Shiga toxin-producing Escherichia (E.) coli (STEC), commensal E. coli and extended-spectrum beta-lactamase- (ESBL) or ampicillinase class C (AmpC) beta-lactamase-producing E. coli (in wild boars, roe deer and wild ducks and geese). In addition, the presence of methicillin-resistant Staphylococcus aureus (MRSA) was investigated in nasal swabs from wild boars. Isolates obtained in the accredited regional state laboratories were submitted to the National Reference Laboratories (NRLs) for confirmation, characterization and phenotypic resistance testing using broth microdilution according to CLSI. AMR was assessed according to epidemiological cut-offs provided by EUCAST. Salmonella spp. were isolated from 13 of 552 (2.4%) tested wild boar fecal samples, but absent in all 101 samples from wild ducks and geese. Nine of the 11 isolates that were submitted to the NRL Salmonella were susceptible to all tested antimicrobial substances. Campylobacter spp. were isolated from four out of 504 (0.8%) roe deer fecal samples, but not from any of the samples from wild ducks and geese. Of the two isolates received in the NRL Campylobacter, neither showed resistance to any of the substances tested. From roe deer, 40.2% of the fecal samples (144 of 358) yielded STEC compared to 6.9% (37 of 536) from wild boars. In wild ducks and geese, no STEC isolates were found. Of 150 STEC isolates received in the NRL (24 from wild boars and 126 from roe deer), only one from each animal species showed resistance. Of the 219 isolates of commensal E. coli from wild boars tested for AMR, 210 were susceptible to all 14 tested substances (95.9%). In roe deer this proportion was even higher (263 of 269, 97.8%), whereas in wild ducks and geese this proportion was lower (41 of 49, 83.7%). Nevertheless, selective isolation of ESBL-/AmpC-producing E. coli yielded 6.5% (36 of 551) positive samples from wild boars, 2.3% (13 of 573) from roe deer and 9.8% (10 of 102) from wild ducks and geese. Among the 25 confirmed ESBL-/AmpC-producing isolates from wild boars, 14 (56.0%) showed resistance up to five classes of substances. This proportion was lower in roe deer (3 of 12, 25%) and higher in wild ducks and geese (7 of 10, 70%). None of the 577 nasal swabs from wild boars yielded MRSA. Results indicate that overall, the prevalence of resistant bacteria from certain wild animals in Germany is low, which may reflect not only the low level of exposure to antimicrobials but also the low level of resistant bacteria in the areas where these animals live and feed. However, despite this low prevalence, the patterns observed in bacteria from the wild animals included in this study are an indicator for specific resistance traits in the environment, including those to highest priority substances such as 3rd generation cephalosporins, fluoroquinolones and colistin. Therefore, also continuous monitoring of the occurrence of such bacteria in wildlife by selective isolation is advisable. Furthermore, the possible role of wildlife as reservoir and disperser of resistant bacteria would need to be assessed, as wild animals, and in particular wild ducks and geese could become spreaders of resistant bacteria given their capacity for long-range movements.

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.

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.

Evaluation of temocillin for phenotypic carbapenemase screening of Escherichia coli and Salmonella enterica isolates in relation to the presence of genes encoding ESBLs and carbapenemase production.

The expression of enzymes of the OXA-48 carbapenemase group is difficult to detect by phenotypic methods owing to frequent low levels of carbapenem resistance and negative results with some screening methods. Temocillin has been shown to be a good option for phenotypic screening as it is hydrolysed by the OXA-48-group enzymes, whereas ESBLs, AmpC and some other carbapenemases have a lower hydrolytic effect on this antimicrobial. However, no epidemiological cut-off for temocillin is available. To evaluate temocillin MICs in relation to the presence or absence of genes encoding ESBLs and carbapenemases in Escherichia coli and Salmonella enterica. In this study, 111 E. coli and 102 S. enterica isolates, including WT and well-characterized ESBL-, AmpC- or carbapenemase-producing isolates, were tested by three independent laboratories. MICs were determined according to the CLSI guidelines by agar dilution with the test range from 0.5 to 512 mg/L temocillin and WGS was performed and analysed with ResFinder. Some overlap was detected between temocillin MICs for WT and ESBL- or AmpC-producing isolates. However, isolates carrying genes encoding carbapenemases showed a broader range of MICs for both E. coli and S. enterica. Higher MICs were observed for the OXA-48 group, VIM and some NDM-producing isolates, whereas isolates harbouring KPC enzymes showed low MICs. The results indicate that temocillin MICs enable phenotypic distinction between strains producing OXA-48-group enzymes and both WT susceptible and ESBL/AmpC-carrying isolates, whereas the distinction from other carbapenemases likely requires genotypic testing.

Rapid antimicrobial susceptibility testing of positive blood cultures by direct inoculation and reading of disc diffusion tests after 3-4 hours.

The aim of the study was to validate whether rapid antimicrobial susceptibility testing (AST) by very early reading of disc diffusion tests would provide reliable results in daily routine work. A total of 264 positive blood culture bottles were examined, of which 178 were examined as part of the daily routine workflow. Enterobacterales were evaluated for resistance to cefotaxime, ceftazidime, gentamicin and ampicillin. Staphylococcus aureus (S.aureus) was tested for resistance to cefoxitin as a marker of methicillin-resistant S.aureus (MRSA). The zones were read after 3h, and if there was insufficient growth, 30 and 60min later. The results were compared to standard overnight AST. For ampicillin, gentamicin and cefoxitin, there were no errors. For cefotaxime, there were three minor (1.5%), three major (1.5%) and no very major errors. For ceftazidime, there were 13 minor (6.5%) errors only. With the exception of one minor error, all errors were ESBL-A- or AmpC-producing isolates where rapid AST showed a higher degree of resistance than standard AST. This low-cost method may contribute to early effective antibacterial treatment by providing reliable results of AST within 3-4h. Special breakpoints for early reading are a prerequisite.

Characterization of Salmonella enterica serovars recovered from meat products legally and illegally imported into the EU reveals the presence of multiresistant and AmpC-producing isolates

BackgroundFood products of animal origin brought into the EU from third countries, both legally and illegally, can harbor foodborne pathogens such as Salmonella enterica. In this study, we examined five S. enterica isolates recovered either from legally imported chicken meat (n = 3) or from meat products confiscated from air travel passengers arriving in Germany (n = 2). The isolates were serotyped and further characterized by antimicrobial susceptibility testing, PCR-detection and sequencing of genes associated with antimicrobial resistances, and macrorestriction analysis. Transferability of resistance to third-generation cephalosporins was assessed by conjugation experiments and the plasmids tested for their incompatibility groups.ResultsThe three isolates from legal imports were identified as S. Heidelberg or as non-flagellated. All three isolates were identified as AmpC producers carrying blaCMY-2 and as non-susceptible to ciprofloxacin. They were additionally resistant to tetracycline and sulfamethoxazole. The blaCMY-2-carrying plasmids were transferable by conjugation and belonged to incompatibility groups IncI1 or IncA/C. The two isolates from illegally imported meat belonged to the serovars Infantis or Weltevreden. The former was phenotypically resistant to five classes of antimicrobial agents while the S. Weltevreden isolate was fully susceptible to all agents tested.ConclusionThe results of this study demonstrate that meat products imported from third countries, both legally and illegally, can harbor multiresistant Salmonella enterica. Consequently, these imports could constitute a source for the dissemination of antimicrobial resistant isolates, including those resistant to third-generation cephalosporins and fluoroquinolones.

Frequency of extended-spectrum β-lactamase (ESBL)– and AmpC β-lactamase–producing Enterobacteriaceae in a cheese production process

The aim of this study was to investigate the extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase activities of Enterobacteriaceae isolated from raw milk and cheese production line and to determine the probability of transmitting these bacteria to consumers. One hundred seventy-three samples from raw milk and cheese production lines were analyzed; 64 isolates were confirmed as Enterobacteriaceae. Sixteen of 64 isolates (25%) were resistant to at least one cephalosporin according to European Committee on Antimicrobial Susceptibility Testing Standards (EUCAST) and Clinical and Laboratory Standards Institute criteria. Seven of the 16 resistant isolates (43.75%) had confirmed ESBL activity. Additionally, phenotypic AmpC β-lactamase activity was observed in 31 (48.44%) of 64 Enterobacteriaceae isolates and confirmed in 27 of the 31 strains (89.1%). Overall, 3 isolates showed both ESBL activity and AmpC resistance, 28 isolates were only AmpC resistant, and 5 isolates had ESBL activity alone. Of the 173 samples, the proportions of samples that contained ESBL- and AmpC-producing isolates were 4.64 and 15.6%, respectively. Five of the ESBL-positive isolates (62.5%) and 11 of the AmpC-positive isolates (40.7%) were obtained from bulk milk tanks; therefore, the bulk tank plays a very important role in the spread of antibiotic-resistant bacteria. Periodic cleaning and maintenance of bulk tanks should be performed and recorded. Effective food safety and hygiene practices should significantly reduce cross-contamination in dairy plants.

Clonal relationships, antimicrobial susceptibilities, and molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates from urinary tract infections and fecal samples in Southeast Iran.

Multidrug-resistant (MDR) Escherichia coli, a species that is a leading cause of urinary tract infections (UTIs) and is a major global public health concern. This study was designed to detect the differences in antibiotic resistance patterns, the production and type of extended spectrum β-lactamases (ESBLs), and the clonal relationships among E. coli isolates from UTIs and fecal samples. Antibacterial resistance was determined by the disk diffusion method. ESBL, carbapenemase, and AmpC-producing isolates were detected phenotypically. Then, the ESBL genes were sequenced to detect the type. Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was performed on the ESBL-positive isolates. The most common effective antibacterial agents were colistin, imipenem, and amikacin. Among the isolates, 204 (56.6%) were MDR. Of the 163 ESBL-positive isolates, 11 (6.7%) produced AmpC, and the frequencies of beta-lactamase-positive genes were as follows: bla CTX-Mgroup1, 76%; bla TEM1, 74.8%; bla SHV12, 1.2%; and bla OXA1, 12.88%. ERIC PCR showed a diverse pattern, suggesting that clonal spread of E. coli in this area is uncommon, and that most of the infecting strains are endogenous. The high rates of antibacterial-resistant and MDR isolates are quite important since these strains can act as source of resistant bacteria that can be spread in the community. Controlling antibiotic use, against inappropriate use and abuse, in the community and continuous surveillance of emerging resistance traits are critical to controlling the spread of resistance.

Genotyping and antimicrobial resistance in Escherichia coli from pig carcasses

ABSTRACT: The increasing antimicrobial resistance observed worldwide in bacteria isolated from human and animals is a matter of extreme concern and has led to the monitoring of antimicrobial resistance in pathogenic and commensal bacteria. The aim of this study was to evaluate the antimicrobial resistance profile of Escherichia coli isolated from pig carcasses and to assess the occurrence of relevant resistance genes. A total of 319 E. coli isolates were tested for antimicrobial susceptibility against different antimicrobial agents. Moreover, the presence of extended-spectrum β-lactamase (ESBL) and inducible ampC-β-lactamase producers was investigated. Eighteen multi-resistant strains were chosen for resistance gene detection and PFGE characterization. The study showed that resistance to antimicrobials is widespread in E. coli isolated from pig carcasses, since 86.2% of the strains were resistant to at least one antimicrobial and 71.5% displayed multi-resistance profiles. No ampC-producing isolates were detected and only one ESBL-producing E. coli was identified. Genes strA (n=15), floR (n=14), aac(3)IVa (n=13), tetB (n=13), sul2 (n=12), tetA (n=11), aph(3)Ia (n=8) and sul3 (n=5) were detected by PCR. PFGE analysis of these multi-resistant E. coli strains showed less than 80% similarity among them. We conclude that antimicrobial multi-resistant E. coli strains are common on pig carcasses and present highly diverse genotypes and resistance phenotypes and genotypes.

Occurrence of extended-spectrum β-lactamase, AmpC and virulence genes in Escherichia coli isolates from vegetable salads in Morocco

PurposeThe purpose of this paper is to assess the occurrence of the extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamase genes in 144 Escherichia coli isolates recovered from 160 vegetable salad samples.Design/methodology/approachAmong the 144 E. coli isolates recovered from 160 vegetable salads, 17 (12 percent) ceftazidime-resistant isolates were screened for ESBL production with the double disk-diffusion test. The ESBL-producing isolates were characterized for antimicrobial resistance, the presence of virulence genes and plasmid-mediated quinolone resistance (PMQR) determinants. The isolates were also subjected to phylogenetic group typing. The existence of plasmid AmpC genes and mutations in the regulatory region of the chromosomal AmpC gene was assessed using polymerase chain reaction (PCR) and sequencing. All β-lactamase isolates were further characterized by pulsed-field gel electrophoresis to determine the genetic relatedness.FindingsOverall, 17 (12 percent) of the 144 E. coli isolates studied were ceftazidime resistant. Among the 17 isolates, 13 (77 percent) were multidrug resistant and four (23.5 percent) were ESBL producers. The bla CTX-M14 was the only gene detected. Of the 12 AmpC-producing isolates, three (18 percent) harbored plasmid-encoded AmpC and sequencing analysis of the chromosomal AmpC genes revealed mutations in the promoter/attenuator region. PMQR determinants were detected in 9 (52 percent) isolates. A was the most prevalent phylogenetic group (56 percent), followed by groups B1 (31 percent), D (6 percent), and B2 (6 percent). PCR showed that six (50 percent) ESBL/AmpC-producing E. coli isolates carried one and/or two virulence genes. Pulsed-field gel electrophoresis showed no epidemiological relationship between these isolates.Originality/valueThis study places vegetable salads within the spectrum of ecological niches that may be vehicles for antibiotic-resistant bacteria/genes with clinical interest and these findings are worthy of attention as their spread to humans by ingestion cannot be dismissed.

Plasmid-mediated resistance to cephalosporins and quinolones in Escherichia coli from American crows in the USA.

American crow (Corvus brachyrhynchos) faeces were tested for Escherichia coli with plasmid-mediated quinolone resistance (PMQR), extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases. A total of 590 faecal samples were collected at four roosting sites in the USA and cultivated on selective media. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed to assess clonality. Transferability of resistance genes was studied using conjugation and transformation bioassays. In total, 78 (13%, n = 590) cefotaxime-resistant isolates were obtained, of which 66 and 12 displayed AmpC and ESBL phenotypes, respectively. Fifty-four AmpC-producing isolates carried blaCMY-2 . Isolates producing ESBLs contained genes blaCTX-M-27 (5 isolates), blaCTX-M-15 (4), blaCTX-M-14 (2) and blaCTX-M-1 (1). Ninety isolates (15%, n = 590) with reduced susceptibility to ciprofloxacin were obtained, among which 14 harboured PMQR genes aac(6')-Ib-cr (4 isolates), qnrB19 (3), qnrS1 (2), qnrA1 (2), qnrB2 (1), qnrB6 (1) and qnrD3 (1). High genetic diversity was revealed by PFGE and MLST. Epidemiologically important E. coli clones (e.g., ST131, ST405) were identified. Plasmids carrying blaCMY-2 were assigned predominantly to IncA/C (8 plasmids), IncI1/ST23 (5) and IncI1/ST12 (3). The study demonstrates a widespread occurrence of E. coli with ESBL, AmpC and PMQR genes associated with clinically important multidrug-resistant clones and epidemic plasmids, in American crows.

Characterisation of extended-spectrum β-lactamase and AmpC β-lactamase-producing Enterobacteriaceae isolated from companion animals in New Zealand

AIMS: To assess the occurrence of, and characterise, extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC)-producing Enterobacteriaceae isolated by veterinary diagnostic laboratories from infection sites in companion animals in New Zealand.METHODS: Selected Enterobacteriaceae isolates were submitted by seven New Zealand veterinary diagnostic laboratories. They were isolated from infection sites in companion animals between June 2012 and June 2013, and were resistant to amoxicillin-clavulanic acid, fluoroquinolones, or any combination of two or more antimicrobials. Based on disk diffusion test results, the isolates were phenotypically categorised according to production of ESBL and AmpC. Genes for ESBL and AmpC production were amplified by PCR and sequenced. Escherichia coli isolates were also typed by multilocus sequence typing.RESULTS: A total of 115 isolates matching the inclusion criteria were obtained from the participating laboratories, of which 74 (64%) originated from dogs and 29 (25%) from cats. Seven bacterial species were identified, of which E. coli was the most common (87/115, 76%). Of the 115 isolates, 10 (9%) expressed the ESBL phenotype, 43 (37%) the AmpC phenotype, and seven (6%) both ESBL and AmpC phenotypes. Of the 60 ESBL and AmpC-producing isolates, 36 (60%) were E. coli. Amongst these isolates, 27/60 (45%) were classified as multidrug resistant, compared with 15/55 (27%) non-ESBL or AmpC-producing isolates (p<0.01). Ninety five isolates were resistant to amoxicillin-clavulanic acid and 58 (61%) of these were ESBL or AmpC-producing. The predominant ESBL genes were blaCTX-M-14 and blaCTX-M-15, and the dominant plasmid-encoded AmpC gene was blaCMY-2. Thirty-eight E. coli multilocus sequence types (ST) were identified, and the most prevalent were ST12 (12/89, 13%), ST131 (6/89, 7%) and ST648 (6/89, 7%). ESBL and AmpC-producing isolates accounted for 35/1,082 (3.2%) of the Enterobacteriaceae isolated by one laboratory network over the study period.CONCLUSIONS AND CLINICAL RELEVANCE: ESBL and AmpC-producing Enterobacteriaceae were associated with clinical infections in companion animals in New Zealand, and were often multidrug resistant. In this study, these organisms accounted for <5% of all Enterobacteriaceae isolated from infection sites by one laboratory network, but their prevalence among isolates resistant to amoxicillin-clavulanic acid was 61%. Therefore routine secondary testing for ESBL and AmpC production by Enterobacteriaceae that are resistant to amoxicillin-clavulanic acid in primary testing could improve the accuracy of definitive antimicrobial therapy in companion animals in New Zealand.