Uncovering the spread of drug-resistant bacteria through next-generation sequencing based surveillance: transmission of extended-spectrum ß-lactamase-producing Enterobacterales by a contaminated duodenoscope.

Contamination of duodenoscopes is a significant concern due to the transmission of multidrug-resistant organisms (MDROs) among patients who undergo endoscopic retrograde cholangiopancreatography (ERCP), resulting in outbreaks worldwide. In July 2020, it was determined that three different patients, all had undergone ERCP with the same duodenoscope, were infected. Two patients were infected with blaCTX-M-15 encoding Citrobacter freundii, one experiencing a bloodstream infection and the other a urinary tract infection, while another patient had a bloodstream infection caused by blaSHV-12 encoding Klebsiella pneumoniae. Molecular characterization of isolates was available as every ESBL-producing isolate undergoes Next-Generation Sequencing (NGS) for comprehensive genomic analysis in our center. After withdrawing the suspected duodenoscope, we initiated comprehensive epidemiological research, encompassing case investigations, along with a thorough duodenoscope investigation. Screening of patients who had undergone ERCP with the implicated duodenoscope, as well as a selection of hospitalized patients who had ERCP with a different duodenoscope during the outbreak period, led to the discovery of three additional cases of colonization in addition to the three infections initially detected. No microorganisms were detected in eight routine culture samples retrieved from the suspected duodenoscope. Only after destructive dismantling of the duodenoscope, the forceps elevator was found to be positive for blaSHV-12 encoding K. pneumoniae which was identical to the isolates detected in three patients. This study highlights the importance of using NGS to monitor the transmission of MDROs and demonstrates that standard cultures may fail to detect contaminated medical equipment such as duodenoscopes.

Dynamic within-host cefiderocol heteroresistance caused by blaSHV-12 amplification in pandrug-resistant and hypervirulent Klebsiella pneumoniae sequence type 11.

AIMS: Although cefiderocol (FDC) is not prescribed in China, FDC-resistant pandrug-resistant hypervirulent Klebsiella pneumoniae (PDR-hvKp) is emerging. In this study, we performed FDC susceptibility testing of clinical Kp isolates to explore the prevalence of FDC-resistant isolates and the mechanism of FDC-resistance. METHODS: We retrospectively selected 151 carbapenem-resistant Kp isolates to assess FDC susceptibility. Seven isolates harboring blaSHV-12 from two patients were enrolled for whole-genome sequencing. The antimicrobial resistance, virulence, blaSHV-12 expression, and fitness costs in different media were examined. The amplification of blaSHV-12 was further investigated by qPCR and long-read sequencing. RESULTS: The 151 isolates showed a low MIC50/MIC90 (1/4 mg/L) of FDC. The seven isolates were ST11 PDR-hvKp, and two represented FDC-resistance (MIC=32 mg/L). The IncR/IncFII plasmids of two FDC-resistant isolates harbored 6 and 15 copies of blaSHV-12, whereas four FDC-susceptible isolates carried one copy and one harbored three copies. These blaSHV-12 genes concatenated together and were located within the same 7.3 kb fragment flanked by IS26, which contributed to the increased expression and FDC resistance without fitness costs. The amplification of blaSHV-12 and FDC resistance could be induced by FDC in vitro and reversed during continuous passage. CONCLUSIONS: The amplification of blaSHV-12 and the consequent dynamic within-host heteroresistance are important concerns for the rational application of antibiotics. Long-read sequencing might be a superior way to detect resistance gene amplification rapidly and accurately.

Emergence of blaSHV-12 and qnrS1 encoded on IncX3 plasmids: Changing epidemiology of extended-spectrum ß-lactamases among Enterobacterales isolated from broilers.

OBJECTIVES: The occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in broilers represents a risk to public health because of the possibility of transmission of ESBL producers and/or blaESBL genes via the food chain or within settings where human-animal interfaces exist. METHODS: This study assessed the occurrence of ESBL producers among faecal samples of broilers at slaughter. Isolates were characterised by multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing. RESULTS: The flock prevalence, determined by sampling crates of 100 poultry flocks, was 21%. The predominant blaESBL gene was blaSHV-12, identified in 92% of the isolates. A variety of Escherichia coli and Klebsiella pneumoniae sequence types (STs) were identified, including extraintestinal pathogenic E. coli ST38, avian pathogenic E. coli ST10, ST93, ST117, and ST155, and nosocomial outbreak clone K. pneumoniae ST20. Whole-genome sequencing was used to characterise a subset of 15 isolates, including 6 E. coli, 4 K. pneumoniae, 1 Klebsiella grimontii, 1 Klebsiella michiganensis, 1 Klebsiella variicola, and 1 Atlantibacter subterranea. Fourteen isolates carried identical or closely related 46338-54929 bp IncX3 plasmids encoding blaSHV-12 and qnrS1. One E. coli isolate carried a 46338 bp IncX3 plasmid, which was integrated chromosomally into ydbD. CONCLUSIONS: The blaSHV-12 gene has replaced the previously predominant blaCTX-M-1 in ESBL-producing Enterobacterales from broilers in Switzerland. Broilers may play a role in the dissemination of blaSHV-12 and qnrS1 associated with epidemic IncX3 plasmids, representing a risk to human and animal health.

Clonal and plasmid-mediated flow of ESBL/AmpC genes in Escherichia coli in a commercial laying hen farm.

Resistance to third- and fourth-generation cephalosporins in Escherichia coli is mainly due to extended-spectrum beta-lactamases (ESBL) and AmpC cephalosporinases, which have been increasingly reported, mainly in isolates from humans and poultry. The aim of this study was to address the flow of antimicrobial resistance determinants in the full laying hen production cycle (four batches followed from day-old chicks to 83/84-week-old layers), using cephalosporin-resistant E. coli as a model and their characterization using whole genome sequencing (WGS). Fifteen out of 22 samples analysed yielded growth on MacConkey agar with cefotaxime (1 mg/L). Of these, 141 isolates were identified as E. coli and 47 were characterized by WGS. Genes detected were three ESBL (blaCTX-M-1 (n = 19); blaCTX-M-14 (n = 1); and blaSHV-12 (n = 9)) and one AmpC (blaCMY-2 (n = 13)). Some isolates only harboured blaTEM-1B (n = 2) or blaTEM-1D (n = 1). IncI1 plasmids were the main platform for ESBL/AmpC genes. In addition, five clones were identified harbouring blaCTX-M-1 (two), blaSHV-12 (one) and blaCMY-2 (two), drawing a clone-plasmid mixed flow model. Gene blaCTX-M-1 was found in the chromosomal DNA of clone 1 over 14 months, and in IncI1/ST3 plasmids over six months; over six months blaSHV-12 was found harboured by clone 3 (IncI1/ST26 plasmids), and 15 months later in a non-replicon detected plasmid. Finally, blaCMY-2 spread for at least 16 months, mainly by IncK2 (including clone 4) and IncI1/ST12 (clone 5) plasmids. Proper use of antimicrobials should be combined with other farm management strategies for the effective control of cephalosporin-resistant E. coli isolates in commercial layer farms.

Antimicrobial Resistance in Escherichia coli from the Broiler Farm Environment, with Detection of SHV-12-Producing Isolates.

Antimicrobial resistance is an important One Health challenge that encompasses the human, animal, and environmental fields. A total of 111 Escherichia coli isolates previously recovered from manure (n = 57) and indoor air (n = 54) samples from a broiler farm were analyzed to determine their phenotypes and genotypes of antimicrobial resistance and integron characterization; in addition, plasmid replicon analysis and molecular typing were performed in extended-spectrum-beta-lactamase (ESBL) producer isolates. A multidrug-resistance phenotype was detected in 46.8% of the isolates, and the highest rates of resistance were found for ampicillin, trimethoprim−sulfamethoxazole, and tetracycline (>40%); moreover, 15 isolates (13.5%) showed susceptibility to all tested antibiotics. None of the isolates showed imipenem and/or cefoxitin resistance. Twenty-three of the one hundred and eleven E. coli isolates (20.7%) were ESBL producers and carried the blaSHV-12 gene; one of these isolates was recovered from the air, and the remaining 22 were from manure samples. Most of ESBL-positive isolates carried the cmlA (n = 23), tet(A) (n = 19), and aac(6')-Ib-cr (n = 11) genes. The following genetic lineages were identified among the ESBL-producing isolates (sequence type-phylogroup-clonotype): ST770-E-CH116−552 (n = 12), ST117-B2-CH45−97 (n = 4), ST68-E-CH26−382/49 (n = 3), ST68-E-CH26−49 (n = 1), and ST10992-A/B1-CH11−23/41/580 (n = 4); the latter two were detected for the first time in the poultry sector. At least two plasmid replicon types were detected in the ESBL-producing E. coli isolates, with IncF, IncF1B, IncK, and IncHI1 being the most frequently found. The following antimicrobial resistance genes were identified among the non-ESBL-producing isolates (number of isolates): blaTEM (58), aac(6')-Ib-cr (6), qnrS (2), aac(3)-II (2), cmlA (6), tet(A)/tet(B) (22), and sul1/2/3 (51). Four different gene-cassette arrays were detected in the variable region of class 1 (dfrA1-aadA1, dfrA12-aadA2, and dfrA12-orf-aadA2-cmlA) and class 2 integrons (sat2-aadA1-orfX). This work reveals the worrying presence of antimicrobial-resistant E. coli in the broiler farm environment, with ESBL-producing isolates of SHV-12 type being extensively disseminated.

Characterization of E. coli Isolates Producing Extended Spectrum Beta-Lactamase SHV-Variants from the Food Chain in Germany.

Resistance of bacteria to 3rd generation cephalosporins mediated by beta-lactamases (ESBL, pAmpC) is a public health concern. In this study, 1517 phenotypically cephalosporin-resistant E. coli were screened for the presence of blaSHV genes. Respective genes were detected in 161 isolates. Majority (91%) were obtained from poultry production and meat. The SHV-12 beta-lactamase was the predominant variant (n = 155), while the remaining isolates exhibited SHV-2 (n = 4) or SHV-2a (n = 2). A subset of the isolates (n = 51) was further characterized by PCR, PFGE, or whole-genome sequencing and bioinformatics analysis. The SHV-12-producing isolates showed low phylogenetic relationships, and dissemination of the blaSHV-12 genes seemed to be mainly driven by horizontal gene transfer. In most of the isolates, blaSHV-12 was located on transferable IncX3 (~43 kb) or IncI1 (~100 kb) plasmids. On IncX3, blaSHV-12 was part of a Tn6 composite transposon located next to a Tn3 transposon, which harbored the fluoroquinolone resistance gene qnrS1. On IncI1 plasmids, blaSHV-12 was located on an incomplete class 1 integron as part of a Tn21 transposon. In conclusion, SHV-12 is widely distributed in German poultry production and spreads via horizontal gene transfer. Consumers are at risk by handling raw poultry meat and should take care in appropriate kitchen hygiene.

Antibacterial potential of Forsythia suspensa polysaccharide against resistant Enterobacter cloacae with SHV-12 extended-spectrum ß-lactamase (ESBL).

In this study, a homogenous polysaccharide (FSP), with an average molecular weight of 9.08 × 104  Da, was isolated from Forsythia suspense and its antibacterial potential against Enterobacter cloacae producing SHV-12 ESBL was investigated. Growth kinetics, in vitro competition and biofilm formation experiments demonstrated that SHV-12 ESBL contributed to a fitness benefit to E cloacae strain. The antibacterial activity of FSP (2.5, 5.0 and 10.0 µg/mL) was tested against E cloacae bearing SHV-12 ESBL gene using bacterial sensitivity, agar bioassay and agar well diffusion assays. It was found that the addition of FSP demonstrated potent antibacterial activities against this bacterial as showed by the decrease of bacterial growth and the increase of the inhibition zone diameter. Furthermore, SHV-12 ESBL gene expression was decreased in E cloacae strain following different FSP treatment in a concentration-dependent manner. In conclusion, these data showed that FSP exhibited potent good antibacterial activity against E cloacae producing SHV-12 ESBL via inhibition of SHV-12 ESBL gene expression, which may promote the development of novel natural antibacterial agents to treat infections caused by this drug-resistant bacterial pathogen.

Characterization of blaNDM-1- and blaSHV-12-Positive IncX3 Plasmid in an Enterobacter Hormaechei New Sequence Type 1000 from China.

PURPOSE: Carbapenem-resistant Enterobacter cloacae complex has been reported worldwide and becomes a new challenge for clinical management. The present study was to characterize the IncX3 plasmid encoding bla NDM-1 and bla SHV-12 gene in E. hormaechei sequence. MATERIALS AND METHODS: EcHK001 was recovered from the sputum sample of a patient. Species identification and antimicrobial susceptibility testing were performed using the VITEK 2 system, while further classification was carried out by hsp60 typing. The presence of NDM-1 was detected by PCR and sequencing. Conjugation experiments and southern blotting were carried out to determine the transferability of the NDM-1-carrying plasmid. Whole-genome sequencing and analysis were conducted to better understand the molecular characteristics of the multi-drug resistant isolate. RESULTS: Strain EcHK001 was classified as E. hormaechei of new sequence type 1000. Multiple drug-resistant genes were detected. The bla NDM-1 and bla SHV-12 genes were located on a self-transferable IncX3 plasmid. Synonymous mutations were identified in the genes encoding TEM-1 and ACT-17. Phylogenetic analysis indicated that EcHK001 clustered into a different clade from domestic strains. CONCLUSION: The rapid spread of the recurrent IncX3 plasmid highlights the need for continuous surveillance of the NDM-1 dissemination. The presence of mutations in existing carbapenem-resistant genes may generate potential new variants and raise serious challenges for clinical treatment.

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

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

Prevalence of Extended-Spectrum ß-Lactamase-Producing Bacteria on Fresh Vegetables in Japan.

Extended-spectrum ß-lactamase (ESBL)-producing bacteria are spreading rapidly, posing a threat to human and animal health. Contamination of vegetables with antimicrobial-resistant bacteria or those harboring antimicrobial resistance genes or a combination of both presents a potential route of transmission to humans. Therefore, the aim of this study was to determine the prevalence of these bacteria in fresh vegetables in Japan. A total of 130 samples of fresh vegetables were collected from seven supermarkets in Japan. The predominant genus detected was Pseudomonas spp., including 10 ESBL-producing strains, isolated from 10 (7.7%) of the vegetable samples. Two ESBL genes were detected, blaTEM-116 (n = 7) and blaSHV-12 (n = 3), and some of these strains were resistant to multiple antibiotics. Because vegetables are often consumed raw, those contaminated with ESBL producers could represent an important route of transmission to humans in Japan. Thus, more stringent hygiene measures and monitoring are required to prevent transmission via this source.

Structural investigation of a polysaccharide from the mycelium of Enterobacter cloacae and its antibacterial activity against extensively drug-resistant E. cloacae producing SHV-12 extended-spectrum ß-lactamase.

In this study, a polysaccharide (ECP) was isolated from the mycelium of Enterobacter cloacae and was found to exhibit strong antibacterial activities against E. cloacae producing SHV-12 ESBL with the increase of the inhibition zone diameter. Its minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 12.5 mg/L and 25 mg/L, respectively. ECP at these concentrations immediately inhibited planktonic growth of the bacteria especially at the time from 2 to 10 h. Flow cytometry analysis further revealed that almost all the bacterial cells were damaged following ECP treatment. The permeability of the cytoplasmic membrane of E. cloacae was increased when ECP concentrations increasing, as evidenced by an influx of Na and an efflux of K, P or S, the leakage of intracellular ATP and the UV-absorbing substances, as well as the depolarization of the cytoplasmic membrane, indicating that bactericidal activity of ECP was achieved by inducing cell membrane damage.

Increased exposure to extended-spectrum ß-lactamase-producing multidrug-resistant Enterobacteriaceae through the consumption of chicken and sushi products.

The aim of this study was to determine the occurrence and patterns of resistance of extended-spectrum ß-lactamase (ESBL) producing Enterobacteriaceae in food products purchased in Navarra, northern Spain. A total of 174 samples of fish and chicken were analyzed from September 2015 to September 2016, including raw and ready-to-eat products: trout (n = 25), salmon (n = 28), panga (n = 13), chicken nuggets and chicken scalopes (n = 32), sushi (n = 31) and sliced cooked poultry (n = 45). Cefpodoxime-resistant strains were isolated on ChromID ESBL agar and further phenotypic (antimicrobial study on MicroScan© NM37 panel) and genotypic characterization (multiplex PCR, sequencing and multi-locus sequence typing, MLST) was performed to confirm and characterize ESBL producers. Raw chicken and sushi have been determined as the most risky products regarding transmission of ESBL-producing Enterobacteriaceae (occurrence 53.1% and 19.4%, respectively), while sliced cooked poultry products appear to be a safe product in this aspect. With regard to raw fish, prevalence in salmon was lower (3.6%) than in trout and panga (16.0%). Ninety-eight per cent of ESBL isolates (n = 50) show multidrug-resistant profiles, highlighting the high resistances against quinolones and tetracyclines observed in chicken isolates, as well as against ertapenem and chloramphenicol in sushi strains. Predominant ß-lactamase type was SHV-12 (50.1%), followed by TEM-type (24.5%) and CTX-M (20.8%). In addition, CTX-M type was only detected in chicken products. The phylogenetic study showed the prevalence of groups A (35%), F (25%) and B1 (15%), usually related to nonvirulent strains. MLST E. coli isolates (n = 20) were grouped into 5 clonal complexes (CC) and 15 sequence types (ST), showing high clonal diversity. ST117 was the prevalent sequence type, while the human pathogen ST131 was not detected in this study. The high prevalence of ESBL-producing multidrug-resistant Enterobacteriaceae detected in products of widespread consumption such as chicken and sushi, increases the concern regarding human exposure to superbugs and encourages the need to improve surveillance of this public health issue.

Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae from Pharmaceutical Wastewaters in South-Western Nigeria.

Emergence and spread of Klebsiella pneumoniae isolates producing extended-spectrum ß-lactamases (ESBLs) present a major threat to public health. In this study, we characterized ß-lactam-resistant K. pneumoniae isolates from six wastewater samples obtained from two pharmaceutical industries located in Lagos and Ogun States, Nigeria. Bacteria were isolated by using MacConkey agar; species identification and antibacterial susceptibility testing were performed by Vitek 2. Etest was used for ESBL phenotype confirmation. The presence of ß-lactamase genes was investigated by PCR and sequencing. Bacterial strain typing was done by XbaI-macrorestriction and subsequent pulsed-field gel electrophoresis (PFGE) as well as multilocus sequence typing (MLST). Thirty-five bacterial species were isolated from the six samples; among them, we identified seven K. pneumoniae isolates with resistance to ß-lactams and co-resistance to fluoroquinolones, aminoglycosides, and folate pathway inhibitors. The ESBL phenotype was confirmed in six K. pneumoniae isolates that harbored ESBL genes blaCTX-M-15 (n = 5), blaSHV-2 (n = 1), and blaSHV-12 (n = 1). PFGE and MLST analysis revealed five clones belonging to four sequence types (ST11, ST15, ST37, ST101), and clone K. pneumoniae-ST101 was present in the wastewater samples from two different pharmaceutical industries. Additionally performed conjugation assays confirmed the location of ß-lactamase genes on conjugative plasmids. This is the first confirmation of K. pneumoniae isolates producing CTX-M-15-ESBL from pharmaceutical wastewaters in Nigeria. The co-resistance observed might be a reflection of the different drugs produced by these industries. Continuous surveillance of the environmental reservoirs of multidrug-resistant bacteria is necessary to prevent their further spread.

High Prevalence of Non-ST131 CTX-M-15-Producing Escherichia coli in Healthy Cattle in Lebanon.

Extended-Spectrum Beta-Lactamases (ESBL)-producing Escherichia coli have disseminated in both humans and animals worldwide. However, the ESBL epidemiology in these two reservoirs differs markedly, with CTX-M-15 frequently found in humans and CTX-M-1 preferentially found in animals. Our goal was to estimate the prevalence of fecal carriage of ESBL producers in cattle from 31 farms in Lebanon and to characterize the responsible enzymes. This prevalence was high (26/31, 84% of ESBL-positive farms), with a majority of isolates producing CTX-M-15 (27/40, 67.5%). Strikingly, this distribution is reminiscent of the human ESBL epidemiology, even though none of the bovine isolates belonged to the ST131 human clone. This is the first report of ESBL-producing E. coli in animals in Lebanon. Our data rather suggest the spread of CTX-M-15 plasmids in different E. coli backgrounds. Nonetheless, some CTX-M-15-producing E. coli clones found here have already been reported from animal, human, or environmental sources.

A Review of SHV Extended-Spectrum ß-Lactamases: Neglected Yet Ubiquitous.

ß-lactamases are the primary cause of resistance to ß-lactams among members of the family Enterobacteriaceae. SHV enzymes have emerged in Enterobacteriaceae causing infections in health care in the last decades of the Twentieth century, and they are now observed in isolates in different epidemiological settings both in human, animal and the environment. Likely originated from a chromosomal penicillinase of Klebsiella pneumoniae, SHV ß-lactamases currently encompass a large number of allelic variants including extended-spectrum ß-lactamases (ESBL), non-ESBL and several not classified variants. SHV enzymes have evolved from a narrow- to an extended-spectrum of hydrolyzing activity, including monobactams and carbapenems, as a result of amino acid changes that altered the configuration around the active site of the ß -lactamases. SHV-ESBLs are usually encoded by self-transmissible plasmids that frequently carry resistance genes to other drug classes and have become widespread throughout the world in several Enterobacteriaceae, emphasizing their clinical significance.

Association of antibiotic resistance with SHV-12 extended-spectrum ß-lactamase in Enterobacter cloacae.

The association between antibiotic resistance and SHV-12 extended-spectrum ß-lactamase (ESBL) in Enterobacter cloacae remains unknown. The aim of the present study was to investigate the prevalence of both chromosome- and plasmid-borne SHV-12 ESBL genes in Enterobacter cloacae. Transmission of the SHV-12 ESBL gene was explored, and the risk factors for antibiotic resistance in E. cloacae were analyzed. Polymerase chain reaction (PCR) results showed that 58 out of the 100 isolates carried the SHV-12 ESBL gene: 34.48% of them occurred in the chromosome, 48.28% were plasmid-borne and 17.24% appeared in both. Enterobacterial repetitive intergenic consensus-PCR tests detected 82 chromosomal genotypes. Conjugation assays showed that 70.00% of plasmid-borne SHV-12 ESBL genes were successfully transconjugated into E. coli C600 and that the antibiotic resistance phenotype of E. cloacae was partially (84%) or completely (10%) transferred. A significantly higher SHV-12 ESBL detection rate was found in patients with underlying conditions and/or complications compared with those without (P<0.05). The detection of SHV-12 ESBL-producing E. cloacae from vertical transmission varied significantly across clinical departments and age groups (P<0.05), with the highest rates in the intensive care unit and the group of patients aged ≥60 years. The present results indicate that the location and transmission efficiency of SHV-12 ESBL are closely correlated with the antibiotic resistance of E. cloacae.

Mechanisms of cephalosporin resistance in indicator Escherichia coli isolated from food animals.

Resistance to ß-lactams is considered one of the major global problems and recently it became the most frequently studied topic in the area of antimicrobial resistance. The study was focused on phenotypic and genetic characterisation of commensal Escherichia coli (E. coli), including those producing cephalosporinases, isolated from gut flora of healthy slaughter animals. E. coli were cultured simultaneously on MacConkey agar (MCA) and cefotaxime supplemented MCA. The isolates were confirmed with ONPG and indol tube tests as well as PCR targeting uspA gene. Microbroth dilution method was applied for determination of Minimal Inhibitory Concentrations and interpreted according to EUCAST epidemiological cut-off values. Cephalosporin resistance phenotypes were defined by E-tests (BioMerieux) and relevant gene amplicons from selected strains were sequenced. A total of 298 E. coli isolates with cephalosporin resistance (ESC) found in 99 ones, were obtained from 318 cloacal or rectal swabs deriving from broilers, layers, turkeys, pigs and cattle. Both extended spectrum ß-lactamase (ESBL) and ampC-cephalosporinase resistance phenotypes were noted in all tested animal species but cattle. At least one of the analysed genes was identified in 90 out of 99 cephalosporin-resistant isolates: blaTEM (n=44), blaCMY (n=38), blaCTX-M (n=33) and blaSHV (n=12). None of the phenotypes was identified in nine isolates. Sequencing of PCR products showed occurrence of ESBL-genes: blaCTX-M-1/-61, blaSHV-12, blaTEM-1,-52/-92,-135 and ampC-gene blaCMY-2. They were located on numerous and diverse plasmids and resistance transferability was proved by electroporation of blaSHV-12 and blaCTX-M-1/-61 located on X1 plasmids. Detection of cephalosporin resistant E. coli confirms the existence of resistance genes reservoir in farm animals and their possible spread (i.e. via IncX1 plasmids) to other bacteria including human and animal pathogens. The identified genetic background indicates on ecological aspects of selection and dissemination of cephalosporin resistance in E. coli isolated from food-producing animals rather than its potential role for public health threats.

Prevalence and diversity of extended-spectrum-ß-lactamase-producing Enterobacteriaceae from marine beach waters.

A total of 1,351 Enterobacteriaceae isolates from 144 seawater samples were collected over a four-year period from three public beaches in the eastern Adriatic Sea in Croatia. Approximately 35% of the strains were multidrug-resistant. BlaESBL genes were detected in 4.2% of the isolated Enterobacteriaceae, the main species of which were Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. BlaTEM-1+SHV-12 was the most dominant genotype, followed by blaCTX-M-15.Raoultella terrigena and E. intermedius simultaneously harboured blaTEM-1,blaSHV-11/12 and blaCTX-M-15. Isolate fingerprinting revealed that marine E. coli isolates were clonally related to CTX-M-producing strains from a regional university hospital. These results indicate that marine beach waters are reservoirs of ESBL-producing Enterobacteriaceae and thus constitute a public health problem with further potential to act as mediators in gene flow between marine coastal areas and clinical settings.

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.

Emergence of CTX-M-9 Extended-Spectrum beta-Lactamase-Producing Enterobacter cloacae Isolates / 대한임상미생물학회지

BACKGROUND: The aim of this study is to assess the prevalence and to investigate the molecular epidemiology of Ambler class A extended-spectrum beta-lactamase (ESBL)-producing Enterobacter cloacae isolates in a university hospital in Busan, Korea. METHODS: Non-duplicated clinical isolates of E.cloacae from patients admitted in Kosin University Gospel Hospital were collected during the period from January through September, 2003. ESBL-production was examined by the double-disk synergy test (DDST) and the transferability of cefotaxime-resistance by conjugation. MICs of beta-lactam antibiotics were determined by the agar dilution method and Ambler class A ESBL genes were searched by PCR amplification. Enterobacterial repetitive intergenic consensus (ERIC) PCR was performed to investigate epidemiological relationships among bla CTX-M-9 gene-carrying E.cloacae isolates. RESULTS: Antimicrobial resistance rates of E.cloacae isolates (n=148) to ceftazidime, cefotaxime, and aztreonam were 50.0%, 29.6%, and 48.0%, respectively. Among 50 E.cloacae isolates intermediate or resistant to more than one expanded-spectrum beta-lactam agent, 41 (27.7%) showed positive results in DDST; of these 41 isolates, 1 was found to carry bla TEM-52 gene, 16 carried bla SHV-12 gene, 4 bla CTX-M-9 gene, and 19 both bla SHV-12 and bla CTX-M-9 genes. The 23 E.cloacae isolates carrying bla CTX-M-9 gene showed 9 different profiles by ERIC PCR. CONCLUSION: ESBL-producing E.cloacae was not uncommon in a university hospital in Busan, Korea. The commonest types of ESBLs produced by E.cloacae isolates were SHV-12 and CTX-M-9. CTX-M-9 ESBL-producing E.cloacae isolates showed diverse ERIC-PCR profiles, indicating that they were not originated from a common source.