Rare Plasmid-Mediated AmpC Beta-Lactamase DHA-1 Located on Easy Mobilized IS26-Related Genetic Element Detected in Escherichia coli from Livestock and Food in Germany.

AmpC beta-lactamases cause resistance to third-generation cephalosporins, including beta-lactamase inhibitors. In Escherichia coli from the German food production chain, the majority of AmpC beta-lactamase activity can be attributed to plasmid-mediated CMY-2 or overproduction of chromosomal AmpC beta-lactamase, but occasionally other enzymes like DHA-1 are involved. This study investigated the prevalence of the AmpC beta-lactamase DHA-1 in ESBL/AmpC-producing E. coli (n = 4706) collected between 2016 and 2021 as part of a German antimicrobial resistance monitoring program along the food chain. Eight isolates (prevalence < 0.2%) were detected and further characterized by PFGE, transformation and conjugation experiments as well as short-read and long-read sequencing. All eight strains harbored blaDHA-1 together with qnrB4, sul1 and mph(A) resistance genes on an IS26 composite transposon on self-transferable IncFII or IncFIA/FIB/II plasmids. During laboratory experiments, activation of the translocatable unit of IS26-bound structures was observed. This was shown by the variability of plasmid sizes in original isolates, transconjugants or transferred plasmids, and correspondingly, duplications of resistance fragments were found in long-read sequencing. This activation could be artificial due to laboratory handling or naturally occurring. Nevertheless, DHA-1 is a rare AmpC beta-lactamase in livestock and food in Germany, and its dissemination will be monitored in the future.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Genomic landscape of the DHA1 family in Candida auris and mapping substrate repertoire of CauMdr1.

The last decade has witnessed the rise of an extremely threatening healthcare-associated multidrug-resistant non-albicans Candida (NAC) species, Candida auris. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the major facilitator superfamily (MFS) of efflux pumps, drug/H+ antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against most of the tested substrates, the ortholog when overexpressed in a minimal background Saccharomyces cerevisiae strain, AD1-8u-, showed significant enhancement in the minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms. KEY POINTS: • Fourteen putative DHA1 transporters are encoded in the Candida auris genome. • Deletion of the CauMDR1 gene does not lead to major changes in drug resistance. • CauMdr1 recognizes and effluxes numerous xenobiotics, including prominent azoles.

First report of two foodborne Salmonella enterica subsp. enterica serovar Bovismorbificans isolates carrying a novel mega-plasmid harboring blaDHA-1 and qnrB4 genes.

Salmonella enterica displaying resistance to extended-spectrum cephalosporins and fluoroquinolone (FQs) has been deemed a high-priority pathogen by the World Health Organization (WHO). While CTX-M type acquired ß-lactamases have been detected in S. enterica serovar Bovismorbificans, DHA enzymes have been rarely reported in S. Bovismorbificans. In this study, we here report for the first time the isolation of two multi-drug resistant (MDR) S. Bovismorbificans strains co-harboring plasmid-encoded AmpC (pAmpC) ß-lactamase gene (blaDHA-1) and qnrB gene, 16Sal017 isolated from a chicken meat sample and 16Sal018 from a grass carp fish sample, collected from retail markets in Guangzhou, China. The blaDHA-1 and qnrB genes in these two strains were both located on the same novel 217,773 bp IncHI2 plasmid belonged to ST2. The plasmid contained 16 additional acquired antimicrobial resistance genes encoding resistance to eight antibiotic classes and quaternary ammonium compound. Besides, 16Sal017 contained an additional 10,124 bp Col (pHAD28)-like plasmid harboring qnrS1. The blaDHA-1 and qnrB4 genes were located in an 18,198 bp region, sul1-qacEΔ1-ampR-blaDHA-1-pspABCDF-qnrB4-sapABC-IS91-sul1-qacEΔ1, which has been identified in various bacteria species, indicating the high transfer ability of blaDHA-1 and qnrB4 genes within this gene cluster. The IncHI2 plasmid was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquiring of multiple resistance in the transconjugants. Genome sequence comparisons by cgMLST and MAUVE alignment indicated 16Sal017 and 16Sal018 are highly similar and are not epidemiologically linked with strains from other sources and countries. Our findings suggest S. Bovismorbificans as a new host for conjugative mega-plasmid harboring blaDHA-1 and qnrB4 genes, and highlight the potential transmission opportunity of these S. Bovismorbificans clones through the food chain, which need continuous investigation.

Deceiving Phenotypic Susceptibility Results on a Klebsiella pneumoniae Blood Isolate Carrying Plasmid-Mediated AmpC Gene blaDHA-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) frequently causes hospital-acquired infections and is associated with high morbidity and mortality. CRKP can have multiple resistance mechanisms and only a few can be routinely detected by commercial molecular or phenotypic assays making surveillance for CRKP particularly challenging. In this report, we identified and characterized an unusual non-carbapenemase-producing CRKP carrying a rare plasmid-borne inducible AmpC gene, blaDHA-1 . The isolate was recovered from blood culture of a 67-year-old female presenting with sepsis post bladder surgery and ureteral stent removal. The primary isolate displayed an indeterminate susceptibility pattern for ceftriaxone by broth microdilution, but was susceptible by disk diffusion with one colony growing within the zone of inhibition. The ceftriaxone resistant colony was sub-cultured and had a minimum inhibitory concentration (MIC) of 2 ug/ml for imipenem (intermediate) and a zone size of 18 mm for ertapenem (resistant), but remained susceptible to cefepime and meropenem. Further phenotypic characterization of this sub-cultured isolate showed carbapenemase activity. Whole genome sequencing (WGS) revealed the presence of two subpopulations of a K. pneumoniae (MLST sequence type 11) from the primary blood culture isolate: one pan-susceptible to beta-lactams tested and the other resistant to the 3rd generation cephalosporins and ertapenem. WGS analysis identified the resistant K. pneumoniae harboring IncFIB(K) and IncR plasmids and the presence of plasmid-borne beta-lactam resistance genes blaOXA-1 and blaDHA-1, an inducible AmpC gene. Additional resistance genes against quinolones (aac(6')-Ib-cr, oqxA, oqB), aminoglycoside (aph(3')-Ia), sulfonamide (sul1), and tetracycline (tet(A)) were also identified. DHA-1 positive K. pneumoniae have been previously identified outside the US, particularly in Asia and Europe, but limited cases have been reported in the United States and may be underrecognized. Our study highlights the importance of using both extended phenotypic testing and WGS to identify emerging resistance mechanisms in clinical Enterobacterales isolates with unusual antimicrobial resistance patterns.

Deadly Puppy Infection Caused by an MDR Escherichia coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6)-Ib-cr - Positive in a Breeding Kennel in Central Italy.

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired ß-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring bla TEM-1b, bla CTX-M-15, bla OXA-1, aph(6)-Ib, aac(6')Ib-cr, aac(3)-Ila, aph(6)-Id, aadA1, qnrB1, sul2, catA1, catB3, tetA, and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, bla DHA-1, qnrB4, mph(A), sul1, and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A bla CMY-2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA, iss, astA, and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6')-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.

Multidrug-Resistant Salmonella Serotype Anatum in Travelers and Seafood from Asia, United States.

A multidrug-resistant Salmonella enterica serotype Anatum strain reported in Taiwan was isolated in the United States from patients and from seafood imported from Asia. Isolates harbored 11 resistance determinants, including quinolone and inducible cephalosporin resistance genes. Most patients had traveled to Asia. These findings underscore the need for global One Health resistance surveillance.

Genomic analysis of two drug-resistant clinical Morganella morganii strains isolated from UTI patients in Pretoria, South Africa.

Morganella morganii is an opportunistic bacterial pathogen of the Enterobacteriaceae family that is occasionally isolated from clinical (animal and human) specimens with varying resistance profiles. Detailed genomic analyses of drug-resistant M. morganii strains are relatively limited, particularly in Africa, which is also due to their relatively low isolation rates from clinical settings. Here we report on two multidrug-resistant clinical M. morganii isolates from urine specimens of two hospitalized patients in South Africa who presented with urinary tract infections in 2013. The isolates, M006 and E042, were only susceptible to carbapenems, amikacin and tigecycline. One strain, M006, had a novel class 1 integron, ln1484, associated with aadA7, sul1and gcuD gene cassettes and a Col3M plasmid replicase gene. The ln1484 intI1:aadA7:sul1 genes were bracketed by a TnAs3 composite transposon while a tet(B) gene was found on an IS4 family transposon. The rare blaDHA-4 and blaDHA-1 AmpC ß-lactamase genes were identified on the isolates' chromosome. The isolates were phylogenetically distant and closely related to other international strains, suggesting that they were not obtained from a single epidemiological source. Further molecular surveillance is necessary to establish the prevalence of these MDR strains in the tertiary hospital. Moreover antibiotic stewardship and antibiotic sensitivity testing of all clinical isolates should be undertaken after empirical treatment to inform tailored therapy as well as reduce escalation of resistance and associated morbidities and mortalities. SIGNIFICANCE AND IMPACT OF THE STUDY: We report on the first clinical Morganella morganii draft genomes from Africa. The isolates were found in the urine of patients presenting with urinary tract infections (UTIs). Notably, they were resistant to important clinical antibiotics, including those used to treat UTIs. Due to the common occurrence of UTIs, particularly among pregnant women for whom drug options are limited, the presence of antibiotic-resistant uropathogens such as M. morganii is a serious public health concern. We therefore characterized the resistance mechanisms and epidemiology of these isolates to provide further insights into their dissemination and background data for future studies.

Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time.

OBJECTIVES: The objective of this work was to provide detailed molecular data on clinically acquired AmpC (qAmpC)-producing Enterobacteriaceae from two different periods (2002-2008 and 2010-2013) in order to clarify the contribution of clonal and plasmid genetic platforms for the current epidemiological scenario concerning extended-spectrum beta-lactams resistance. METHODS: We analysed 1246 Enterobacteriaceae non-susceptible to third-generation cephalosporins from two hospitals and one community laboratory between 2010 and 2013. Bacterial identification, antibiotic susceptibility, identification of qAmpC and plasmid-mediated quinolone resistance genes, clonal (pulsed-field gel electrophoresis (PFGE), Multilocus sequence typing (MLST)) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed by standard methods. Whole-genome sequencing (WGS) was performed in two ST11-Klebsiella pneumoniae isolates harbouring DHA-1. RESULTS: The occurrence of qAmpC was lower (2.6%) than that observed in a previous survey (7.4%), and varied slightly over time. Isolates produced DHA-1 (53%), CMY-2 (44%) or DHA-6 (3%), but significant epidemiological changes were observed in the two surveys. While DHA-1 persisted in different institutions by selection of a worldwide epidemic IncR plasmid in an ST11 harbouring KL105, CMY-2 rates increased over time linked to IncI1 plasmids (instead of IncK or IncA/C2) in multiple Escherichia coli clones. CONCLUSIONS: The higher frequency of DHA-1 qAmpC in these species contrasts with the scenario in most European countries. Furthermore, the different genetic backgrounds associated with either extended-spectrum ß-lactamases (ESBLs) or acquired AmpC ß-lactamases (qAmpC) in our country might have contributed to their differential expansion.

First Report of DHA-1 Producing Enterobacter cloacae Complex Isolate in Bulgaria.

THE AIM: of the present study was to reveal the characteristics of an. MATERIALS AND METHODS: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out. RESULTS: Of 176. CONCLUSIONS: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing.

Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H+ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.

Impact of Inducible blaDHA-1 on Susceptibility of Klebsiella pneumoniae Clinical Isolates to LYS228 and Identification of Chromosomal mpl and ampD Mutations Mediating Upregulation of Plasmid-Borne blaDHA-1 Expression.

Twenty-three Klebsiella pneumoniae (blaDHA-1) clinical isolates exhibited a range of susceptibilities to LYS228, with MICs of ≥8 µg/ml for 9 of these. Mutants with decreased susceptibility to LYS228 and upregulated expression of blaDHA-1 were selected from representative isolates. These had mutations in the chromosomal peptidoglycan recycling gene mpl or ampD Preexisting mpl mutations were also found in some of the clinical isolates examined, and these had strongly upregulated expression of blaDHA-1.

Plasmids carrying DHA-1 ß-lactamases.

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. BlaDHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum ß-lactamases (blaCTX-M-, bla SHV -types), oxacillinases (blaOXA-1, blaOXA-30), penicillinases (bla TEM -type), carbapenemases (bla OXA48 , blaKPC-2), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6'-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.

Stepwise Functional Evolution in a Fungal Sugar Transporter Family.

Sugar transport is of the utmost importance for most cells and is central to a wide range of applied fields. However, despite the straightforward in silico assignment of many novel transporters, including sugar porters, to existing families, their exact biological role and evolutionary trajectory often remain unclear, mainly because biochemical characterization of membrane proteins is inherently challenging, but also owing to their uncommonly turbulent evolutionary histories. In addition, many important shifts in membrane carrier function are apparently ancient, which further limits our ability to reconstruct evolutionary trajectories in a reliable manner. Here, we circumvented some of these obstacles by examining the relatively recent emergence of a unique family of fungal sugar facilitators, related to drug antiporters. The former transporters, named Ffz, were previously shown to be required for fructophilic metabolism in yeasts. We first exploited the wealth of fungal genomic data available to define a comprehensive but well-delimited family of Ffz-like transporters, showing that they are only present in Dikarya. Subsequently, a combination of phylogenetic analyses and in vivo functional characterization was used to retrace important changes in function, while highlighting the evolutionary events that are most likely to have determined extant distribution of the gene, such as horizontal gene transfers (HGTs). One such HGT event is proposed to have set the stage for the onset of fructophilic metabolism in yeasts, a trait that according to our results may be the metabolic hallmark of close to 100 yeast species that thrive in sugar rich environments.

High prevalence of CTX-M-15 and first report of CTX-M-3, CTX-M-22, CTX-M-28 and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae causing urinary tract infections in Bosnia and Herzegovina in hospital and community settings.

AIM: To investigate molecular epidemiology of extended-spectrum ß-lactamase/ESBL and plasmid-mediated AmpC ß-lactamase/pAmpC producing Gram-negative bacteria causing urinary tract infections (UTIs) in Zenica-Doboj Canton, Bosnia and Herzegovina, in the period Decembar 2009-May 2010. METHODS: Antibiotic susceptibility was determined by disc diffusion and broth microdilution according to CLSI guidelines. Double-disk synergy test was performed in order to screen for ESBLs/pAmpC beta-lactamases. PCR was used to detect bla(ESBL)/bla(ampC)/bla(carb) genes. Genetic relatedness of the strains was determined by pulsed-field-gel-electrophoresis (PFGE). RESULTS: Among 85 patients with UTIs caused by ESBL producing isolates, 44 (51.8%) were from in-patients and 41 (48.2%) from outpatients. Klebsiella spp. was the most frequently isolated from in-patients, in 28 (63.6%) cases. Among outpatients, Klebsiella spp./Escherichia coli were the most frequently isolated, in 19 (46.3%)/16 (39.0%) cases. Twenty-one (75.0%) from hospital and nine (47.4%) from outpatient Klebsiella spp. isolates were positive for blaTEM, whereas 27 (96.4%) from in-patients and 6 (31.6%) from outpatient were bla(CTX-M) positive (18 hospital and five outpatient isolates were encoding bla(CTX-M-15)). One Klebsiella oxytoca and one Enterobacter cloacae inpatient isolates were positive for blaCTX-M-28. One Klebsiella pneumoniae outpatient isolate were positive for bla(CTX-M-22) and one E. coli for bla(CTX-M-3). One hospital Proteus mirabilis strain was positive for bla(CMY-2) and two Klebsiella spp. strains for blaDHA-1, whereas two E. coli, one K. oxytoca and one Proteus vulgaris outpatient strains were positive for bla(CMY-2). CONCLUSION: Identification of bla(CTX-M-3), bla(CTX-M-22) and bla(CTX-M-28) among Enterobacteriaceae is uncommon. In this study we report the emergency of CMY-2 and DHA-1 plasmid-mediated beta-lactamases.

Molecular characterization of DHA-1-producing Klebsiella pneumoniae isolates collected during a 4-year period in an intensive care unit.

Seventeen Klebsiella pneumoniae isolates producing DHA-1 ß-lactamase were collected in an intensive care unit between 2006 and 2010. Molecular analysis revealed the predominance of ST48 and ST1263 clones of K. pneumoniae and the spread of DHA-1-encoding plasmids belonging to incompatibility group IncL/M or IncHI2.

An Increase in the clinical isolation of acquired AmpC ß-lactamase-producing Klebsiella pneumoniae in Korea from 2007 to 2010.

We investigated the occurrence and genetic basis of AmpC ß-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum ß-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 µg/mL, respectively. The MIC values for carbapenem were ≥2 µg/mL for 2 K. pneumoniae isolates, both of which carried the bla DHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.

An Increase in the Clinical Isolation of Acquired AmpC beta-Lactamase-Producing Klebsiella pneumoniae in Korea from 2007 to 2010

We investigated the occurrence and genetic basis of AmpC beta-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum beta-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and or =2 microg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.

First Outbreak of Multidrug-Resistant Klebsiella pneumoniae Producing both SHV-12-Type Extended-Spectrum beta-Lactamase and DHA-1-Type AmpC beta-Lactamase at a Korean Hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microngram/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microngram/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.

Genotype distribution of extended-spectrum and AmpC ?-lactamases produced by Escherichia coli and Klebsiella pneumoniae in 10 teaching hospitals of China / 中国感染与化疗杂志

Objective To investigate the genotype distribution of extended-spectrum?-lactamases(ESBLs) and AmpC?-lacta- mases produced by Escherichia coli and Klebsiella pneumoniae in 10 teaching hospitals of China.Methods 90 clinical strains of E.coli and 61 strains of K.pneumoniae isolated in 2003 and confirmed to produce ESBLs were collected from 10 teaching hos- pitals in China.Analytical isoelectric focusing was used to measure the pI of the?-lactamases.Conjugation experiment was used to study the transfer of cefoxitin resistance.Plasmid-mediated AmpC enzyme genes were amplified and sequenced by multiplex polymerase chain reaction (MPCR).Results The prevalence of ESBL-producing E.coli and K.pneumoniae was about 50% in Wuhan,Nanjing and Jinan.The prevalence of ESBL-producing E.coli was lower than K.pneumoniae in Beijing.However,in other hospitals the prevalence of ESBL-producing E.coli was a little higher than K.pneumoniae.About 24.4% of ESBL-pro- ducing E.coli isolates and 19.4% of ESBL-producing K.pneumoniae isolates were resistant to cefoxitin.Cefoxitin-resistant i solate was identified in all hospitals except Shenyang.Major genotype of ESBL-producing isolates was CTX-M.The CTX-M-9 group was the most common group,followed by CTX-M-1.More K.pneumoniae isolates produced both ESBLs and AmpC en- zyme than E.coli.The genotype was CTX-M/DHA-1.The PCR results of 3 transconjugants producing both ESBLs and AmpC enzyme were the same as their donor isolates.Conclusions The genotype of ESBL-producing isolates is mainly CTX-M-9 group in these teaching hospitals.More K.pneumoniae isolates produced both ESBLs and AmpC enzyme than E.coli.Most of these isolates are due to geno type CTX-M/DHA-1,which can spread through plasmid.