Aminoglycoside resistance determinants in multiresistant Escherichia coli and Klebsiella pneumoniae clinical isolates from Turkish and Syrian patients.

Escherichia coli and Klebsiella pneumoniae are frequently found resistance to aminoglycosides in Turkey. The aim of this study was to investigate aminoglycoside resistance in clinical isolates of E. coli and K. pneumoniae from Turkey using both phenotypic and genotypic methods and screening for the prevalence of gene coding for common aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase genes. A total of 88 consecutive, non-duplicated E. coli (n = 65) and K. pneumoniae (n = 23) isolates showing resistance or intermediate resistance to amikacin and/or gentamicin were collected between October 2013 and May 2015 from clinical samples received at Gaziantep Dr. Ersin Arslan Training and Research Hospital. Seventeen isolates were obtained from Syrian patients. Isolates resistant to any of the two aminoglycosides were tested by PCR for seven AME genes, and 22 isolates with amikacin MIC ≥16 mg/L were also tested for 16S rRNA methylase genes. In E. coli isolates, the most frequent genes were aac(6')-Ib (50 strains; 76.9%) and aac(3)-IIa (40 strains; 70.7%), followed by aph(3')-Ia (5 strains; 7.6%) and ant(2″)-Ia (2 strains; 3.1%). Among the 23 resistant K. pneumoniae isolates, the most prevalent gene was aac(3')-IIa (87.0%) followed by aac(6')-Ib (73.9%) and aph(3')-Ia (8.6%). The rmtC gene was detected in one K. pneumoniae isolate. Resistance to aminoglycosides in clinical isolates of E. coli and K. pneumoniae from our center is predominantly caused by AAC(6')-Ib and AAC(3)-II enzymes, while the occurrence of 16S rRNA methylases is so far limited.

Genomics and Susceptibility Profiles of Extensively Drug-Resistant Pseudomonas aeruginosa Isolates from Spain.

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the ß-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in ß-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

Activity of ceftazidime-avibactam against multidrug-resistance Enterobacteriaceae expressing combined mechanisms of resistance.

INTRODUCTION: Antimicrobial resistance in Enterobacteriaceae is increasing worldwide and is making treating infections caused by multidrug-resistant Enterobacteriaceae a challenge. The use of ß-lactam agents is compromised by microorganisms harboring extended-spectrum ß-lactamases (ESBLs) and other mechanisms of resistance. Avibactam is a non ß-lactam agent that inhibits clinically relevant ß-lactamases, such as ESBL and AmpC. The ceftazidime-avibactam combination (CAZ-AVI) was recently approved for use in certain complicated infections, and may provide a therapeutic alternative for infections caused by these microorganisms. METHODS: The in vitro activity of CAZ and CAZ-AVI (AVI at a fixed concentration of 4mg/L) was tested against 250 clinical isolates of Enterobacteriaceae using broth microdilution. EUCAST breakpoint criteria were used for CAZ, and FDA criteria for CAZ-AVI. Clinical isolates included bacteria producing extended-spectrum ß-lactamases (ESBLs) and acquired AmpC ß-lactamases (AACBLs). Porin loss in Klebsiella pneumoniae was also evaluated. RESULTS: The combination of AVI with CAZ displayed excellent activity against clinical isolates of ESBL-producing Escherichia coli and Klebsiella pneumoniae, rendering all the ceftazidime-resistant isolates susceptible to ceftazidime. CAZ-AVI retained activity against porin-deficient isolates of K. pneumoniae producing ESBLs, AACBLs, or both, although MIC values were higher compared to porin-expressing isolates. CAZ-AVI rendered all the ceftazidime-resistant AACBL-producing Enterobacteriaceae tested susceptible to ceftazidime. CONCLUSION: CAZ-AVI showed potent in vitro activity against clinical isolates of Enterobacteriaceae producing ESBLs and/or AACBLs, including K. pneumoniae with loss of porins.

Isolation of VIM-2-producing Pseudomonas monteilii clinical strains disseminated in a tertiary hospital in northern Spain.

We describe here the occurrence of blaVIM-2 in 10 carbapenem-resistant Pseudomonas monteilii strains isolated from different clinical samples from patients at the University Hospital Marqués de Valdecilla in northern Spain. All the blaVIM-2-harboring P. monteilii isolates possessed a class 1 integron, with the cassette array [intI1_blaVIM-2_aac(6')-Ib_qacEΔ1_sul1]. Our results show the emergence of VIM-2-producing multidrug-resistant species other than Pseudomonas aeruginosa or Pseudomonas putida in a Spanish hospital. P. monteilii, although sporadically isolated, should also be considered an important metallo-ß-lactamase (MBL) reservoir.

Prospective multicenter study of carbapenemase-producing Enterobacteriaceae from 83 hospitals in Spain reveals high in vitro susceptibility to colistin and meropenem.

The aim of this study was to determine the impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceae using the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined for Klebsiella pneumoniae and Escherichia coli. A total of 702 Enterobacteriaceae isolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae (74.4%), Enterobacter cloacae (10.3%), and E. coli (8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 for K. pneumoniae and ST131 for E. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. For K. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producing K. pneumoniae (e.g., ST11 and ST405). The dissemination of OXA-48-producing E. coli is a new finding of public health concern. According to the susceptibilities determined in vitro, most of the CPE (94.5%) had three or more options for antibiotic treatment.

Epidemiology of Aeromonas spp. isolated from stool in a tertiary hospital in Cantabria, Northern Spain, in the last five years.

INTRODUCTION: The role of Aeromonas species in gastrointestinal disease is controversial. The aim of this study was to know the epidemiological distribution of Aeromonas spp. isolated from stool in our health area, determine the existence of diarrhea as a significant symptom, identification of existing species in our environment and association as co-pathogen. METHODS: It was a retrospective descriptive study of isolates of Aeromonas spp. in feces (2016-2020). The protocol for these isolates included coproculture, identification by MALDI-TOF (Vitek-MS®, BioMerieux) and confirmation by multiplex PCR. RESULTS: A total of 366 Aeromonas spp. isolates were analyzed being Aeromonas caviae the most prevalent species (289, 78.7%). A total of 58 (15.8%) co-infections were identified, being more frequent in pediatric age (49;84.5%) (p=0.01) and mostly associated with Campylobacter spp. DISCUSSION: Aeromonas spp. prove to be a gastrointestinal pathogen more frequently associated with co-infections in pediatric age, evidencing its appearance especially with Campylobacter spp.

Virulence genes of Aeromonas spp. isolates from stool in Spain.

INTRODUCTION: The role of Aeromonas species in gastrointestinal disease is controversial. The aim was to analyze not only the virulence genes between different species of Aeromonas isolated from feces, but the distribution of these virulence genes between enterotoxigenic strains and co-pathogen strains. METHODS: Retrospective study of isolates of Aeromonas spp. in feces (2016-2021). The protocol included coproculture, identification by MALDI-TOF and confirmation by multiplex PCR. SPSS Statistics program was used. RESULTS: A total of 288 strains were studied for the virulence genes between different species of Aeromonas. To compare virulence genes between Aeromonas as co-pathogen and those isolated alone, 218 strains of the global set were used; 52 as co-pathogens compared with 166 Aeromonas without associated pathogen as controls. CONCLUSIONS: We found no significant differences in the distribution of virulence genes versus co-existence of co-pathogens or not. A. hydrophila is the potentially most virulent species of our set.

Chronic Diarrhea Due to Aeromonas hydrophila in an Immunosuppressed Patient with a Pancreas-Kidney Transplant.

The genus Aeromonas belongs to the Aeromonadaceae family. A patient with a pancreas-kidney transplant had multiple episodes of abdominal sepsis after surgery. Aeromonas hydrophila was isolated in the ascitic and biliary fluid drains. After discharge, the patient had several diarrhea episodes, and A. hydrophila was isolated in four stool samples. We decided to test whether the one strain that we initially isolated in ascitic fluid was the same that appeared in the successive stool samples. Five isolates of A. hydrophila were found in the patient. Identification was performed using the MALDI-TOF system and confirmed via multiplex PCR. The analysis of the REP-PCR fingerprint patterns showed one cluster and confirmed that all isolates were related. We also demonstrated the virulent character of this species associated with genes encoding different toxins (act, alt, ast, hlyA, and aerA). The virulence of this species is associated with the expression of genes that encode different toxins, structural proteins, and metal-associated proteins. This case report highlights the severity of this disease, especially in immunocompromised patients, and its adequate treatment.

In vitro activity of ceftobiprole and dalbavancin against a collection of coagulase-negative staphylococci isolates from clinical samples with reduced susceptibility to daptomycin and/or resistant to linezolid or glycopeptides.

INTRODUCTION: The aim was to investigate the in vitro activity of ceftobiprole and dalbavancin against a collection of coagulase-negative staphylococci (CoNS) isolates with reduced susceptibility to daptomycin or resistant to linezolid and/or glycopeptides. METHODS: A total of 228 CoNS were tested using the Vitek-2 AST-626 cards (bioMérieux) and MIC of daptomycin, linezolid, vancomycin and teicoplanin were confirmed by Etest Strips (bioMérieux). Susceptibility testing for ceftobiprole and dalbavancin were performed by CLSI broth microdilution methodology. Results were interpreted according to 2021 EUCAST clinical breakpoints. RESULTS: Ceftobiprole and dalbavancin were active against 96.0% and 93.0% of CoNS, respectively, MIC90 were 2 and 0.125mg/L. MICs of ceptobiprole were higher against S. hominis and S. haemolyticus (MIC90 4mg/L). Dalbavancin exhibited higher MICs against S. haemolyticus and CoNS with reduced susceptibility to daptomycin and resistant to teicoplanin. CONCLUSION: Ceftobiprole and dalbavancin demonstrated a high in vitro activity against our collection of CoNS isolates.

Outbreak of Arcobacter butzleri? An emerging enteropathogen.

BACKGROUND: Arcobacter butzleri is a gram-negative rod, with microaerobic growth at an optimal temperature of 37°C. It was reported to be the fourth most common Campylobacter-like organism isolated from patients with diarrhoea. OBJECTIVE: Characterise a potential outbreak of A. butzleri detected in a short period of time in the University Hospital Marqués de Valdecilla. METHODS: Eight strains of A. butzleri were detected in our hospital in only two months. Isolates were identified by MALDI-TOF MS system and 16S rDNA sequencing. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and Pulsed Field Gel Electrophoresis (PFGE) were carried out to assess clonal relationship. Gradient strips (Etest) were used to determine susceptibility by agar diffusion. RESULTS: ERIC-PCR and PFGE confirmed the lack of clonal relationship between strains. Erythromycin or ciprofloxacin might be appropriate for antibiotic treatment of infections. CONCLUSIONS: A. butzleri is an emerging pathogen with increasing incidence, and may be underestimated.

Molecular characterization of multidrug resistant Enterobacterales strains isolated from liver and kidney transplant recipients in Spain.

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-ß-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n = 142), liver (n = 98) or kidney/pancreas (n = 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4-6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended-spectrum ß-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4-6 weeks post-transplantation. E. coli producing blaCTX-M-G1 and K. pneumoniae harbouring blaOXA-48 alone or with blaCTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients.

Oral decontamination with colistin plus neomycin in solid organ transplant recipients colonized by multidrug-resistant Enterobacterales: a multicentre, randomized, controlled, open-label, parallel-group clinical trial.

OBJECTIVES: To evaluate the efficacy of oral colistin-neomycin in preventing multidrug-resistant Enterobacterales (MDR-E) infections in solid organ transplant (SOT) recipients. METHODS: Multicentre, open-label, parallel-group, controlled trial with balanced (1:1) randomization in five transplant units. SOT recipients were screened for MDR-E intestinal colonization (extended-spectrum ß-lactamase or carbapenemase producing) before transplantation and +7 and + 14 days after transplantation and assigned 1:1 to receive treatment with colistin sulfate plus neomycin sulfate for 14 days (decolonization treatment (DT) group) or no treatment (no decolonization treatment (NDT) group). The primary outcome was diagnosis of an MDR-E infection. Safety outcomes were appearance of adverse effects, mainly diarrhoea, rash, nausea and vomiting. Patients were monitored weekly until 30 days after treatment. Intention-to-treat analysis was performed. RESULTS: MDR-E rectal colonization was assessed in 768 SOT recipients; 105 colonized patients were included in the clinical trial, 53 receiving DT and 52 NDT. No significant decrease in the risk of infection by MDR-E was observed in the DT group (9.4%, 5/53) compared to the NDT group (13.5%, 7/52) (relative risk 0.70; 95% confidence interval 0.24-2.08; p 0.517). Four patients (5.6%), three (5.6%) in the DT group and one (1.9%) in the NDT group, developed colistin resistance. Twelve patients (22.7%) in the DT group had diarrhoea, eight related to treatment (15.0%); one patient (1.8%) developed skin rash and another (1.8%) nausea and vomiting. Two patients (3.8%) in the NDT group developed diarrhoea. CONCLUSIONS: DT does not reduce MDR-E infections in SOT. Colistin resistance and adverse effects such as diarrhoea are a potential issue that must be taken seriously.

High-risk clones and novel sequence type ST4497 of Klebsiella pneumoniae clinical isolates producing different alleles of NDM-type and other carbapenemases from a single tertiary-care centre in Egypt.

Enterobacteria producing NDM carbapenemases represent a severe diagnostic and therapeutic challenge in healthcare settings. Infections caused by NDM-positive strains are usually associated with high mortality rates and very limited treatment options. A total number of 33 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were included in this study, comprising 30 recovered from clinical diagnostic samples and 3 cultured from screening rectal swabs taken at patient admission. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and antibiotic susceptibility testing was performed by reference broth microdilution and a commercial automated method. Isolates were investigated for carbapenemase production using the ß-CARBA test, the modified carbapenem inactivation method (mCIM) and, for the 30 clinical isolates, by MALDI-TOF/MS, using the MBT STARⓇ-Carba IVD Kit. Carbapenem resistance genes were characterised by PCR and sequencing. Seven different blaNDM gene variants were identified in 94% of the isolates, whilst three variants of blaOXA-48-like were detected in 27% of the isolates. Most CRKP corresponded to high-risk clones (ST147, ST11 and ST15). Novel ST4497 is reported for the first time in this study as well as the first emergence of K. pneumoniae ST231 producing OXA-232 in Egypt. These results indicate an ongoing evolution of the blaNDM genes in our area and confirm the need for a maintained surveillance system in order to monitor the spread of these mobile blaNDM genes.

Antimicrobial Susceptibility and Characterization of Resistance Mechanisms of Corynebacterium urealyticum Clinical Isolates.

Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterial, most frequently involved in urinary tract infections. Most of the C. urealyticum clinical isolates are frequently resistant to several antibiotics. We investigated the susceptibility of 40 C. urealyticum isolated in our institution during the period 2005-2017 to eight compounds representative of the main clinically relevant classes of antimicrobial agents. Antimicrobial susceptibility was determined by the Epsilometer test. Resistance genes were searched by PCR. All strains were susceptible to vancomycin whereas linezolid and rifampicin also showed good activity (MICs90 = 1 and 0.4 mg/L, respectively). Almost all isolates (39/40, 97.5%) were multidrug resistant. The highest resistance rate was observed for ampicillin (100%), followed by erythromycin (95%) and levofloxacin (95%). Ampicillin resistance was associated with the presence of the blaA gene, encoding a class A ß-lactamase. The two rifampicin-resistant strains showed point mutations driving amino acid replacements in conserved residues of RNA polymerase subunit ß (RpoB). Tetracycline resistance was due to an efflux-mediated mechanism. Thirty-nine PFGE patterns were identified among the 40 C. urealyticum, indicating that they were not clonally related, but producing sporadic infections. These findings raise the need of maintaining surveillance strategies among this multidrug resistant pathogen.

Whole-genome sequencing reveals misidentification of a multidrug-resistant urine clinical isolate as Corynebacterium urealyticum.

OBJECTIVES: Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterium associated with urinary tract infections. Most clinical C. urealyticum isolates are multidrug-resistant. Whole-genome sequencing (WGS) of C. urealyticum VH4248 isolated from a clinical urine sample at Hospital Universitario Marqués de Valdecilla, Santander, Spain, was performed to predict its antimicrobial resistance profile and to compare it with results of culture-based phenotypic antimicrobial susceptibility testing. METHODS: Classical microbiological methods and VITEK® MS were used for isolation and initial identification of strain VH4248. Draft genome sequencing was performed on an Illumina HiSeq 2500 platform, followed by assembly and annotation using SPAdes and RAST. Resistance genes were identified through PATRIC, the Pathosystems Resource Integration Center. Average nucleotide identity (ANI) analysis was done using the EDGAR and OrthoANI databases. Antimicrobial susceptibility was determined by Etest. RESULTS: Isolate VH4248 was initially identified asC. urealyticum. Its genome size is 2 261 231 bp with 64.4% GC content. Genome-based identification tools showed an average 93.7% similarity between VH4248 and C. urealyticum genomes deposited in public databases. Therefore, this isolate must be classified as Corynebacterium sp. The blaA and ermX genes as well as a class 1 integron including the aadB and sul1 genes are present in the VH4248 genome. This isolate is highly resistant to ampicillin, erythromycin and trimethoprim/sulfamethoxazole, and moderately resistant to gentamicin and kanamycin. CONCLUSIONS: WGS is a powerful tool forCorynebacterium identification to species level and for detection of unusual resistance determinants, such as that encoded by the class 1 integron in isolate VH4248.

Adherence to Human Colon Cells by Multidrug Resistant Enterobacterales Strains Isolated From Solid Organ Transplant Recipients With a Focus on Citrobacter freundii.

Enterobacteria species are common causes of hospital-acquired infections, which are associated with high morbidity and mortality rates. Immunocompromised patients such as solid organ transplant (SOT) recipients are especially at risk because they are frequently exposed to antibiotics in the course of their treatments. In this work, we used a collection of 106 Escherichia coli, 78 Klebsiella pneumoniae, 25 Enterobacter spp., and 24 Citrobacter spp. multidrug resistant strains isolated from transplant patients (hepatic, renal or renal/pancreatic) in order to examine their ability to adhere in vitro to HT-29 human colon cells, and to determine if some adhesive characteristics are associated with prevalence and persistence of these strains. A total of 33 E. coli (31%), 21 K. pneumoniae (27%), 7 Enterobacter spp. (28%), and 5 Citrobacter spp. (21%), adhered to the colon epithelial cells. Two main adherence patterns were observed in the four species analyzed, diffuse adherence, and aggregative adherence. Under transmission electronic microscopy (TEM), most bacteria lacked visible fimbria on their surface, despite their strong adherence to epithelial cells. None of the strains studied was able to induce any cytotoxic effect on HT-29 cells although some of them strongly colonizing both cells and glass coverslips at high density. Some of the strains failed to adhere to the epithelial cells but adhered strongly to the cover-slide, which shows that microscopy studies are mandatory to elucidate the adherence of bacteria to epithelial cells in vitro, and that quantitative assays using colony forming unit (CFUs) counting need to be supplemented with pictures to determine definitively if a bacterial strain adheres or not to animal cells in vitro. We report here, for the first time, the aggregative adherence pattern of two multidrug resistant (MDR) Citrobacter freundii strains isolated from human patients; importantly, biofilm formation in Citrobacter is totally dependent on the temperature; strong biofilms were formed at room temperature (RT) but not at 37°C, which can play an important role in the colonization of hospital surfaces. In conclusion, our results show that there is a great variety of adhesion phenotypes in multidrug-resistant strains that colonize transplanted patients.

Author Correction: Biofilm formation by multidrug resistant Enterobacteriaceae strains isolated from solid organ transplant recipients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.