Two episodes of bacteremia of zoonotic origin caused by different Streptococcus canis isolates in the same patient within a time span of 1 year.

Two episodes of bacteremia of cutaneous origin in a female patient were caused by two unrelated Streptococcus canis isolates within 1-year interval between the two infection episodes. The most likelihood transmission route in both episodes was a dog pet that habitually licked patient´s legs. Isolates were characterised by antimicrobial susceptibility test and whole genome sequencing. They belonged to sequence type (ST) 40 and 43, respectively. The ST40 isolate harboured antimicrobial resistance genes aadE, ermB and tetO, displaying resistance to erythromycin, clindamycin and tetracyclines, while ST43 isolate did not presented any known antimicrobial resistance determinant and was susceptible to all antibiotics tested. S. canis infections are rare in human; however, attention is needed for patients at risk with companion animals.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

PURPOSE: To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. METHODS: CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. RESULTS: A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. CONCLUSIONS: The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt.

The increasing rates of antimicrobial resistance among carbapenem-resistant Acinetobacter baumannii in the Middle East and North Africa are one of the major concerns for healthcare settings. We characterised the first A. baumannii isolate harbouring five ß-lactamases identified in Egypt. The isolate Ale25 was obtained from an ICU patient of a hospital from Alexandria. The isolate was phenotypically and genotypically screened for carbapenemase genes. The isolate was resistant to carbapenems, aminoglycosides, fluoroquinolones and cefiderocol. Whole-Genome Sequencing identified five ß-lactamase genes, blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57, together with other antibiotic resistance genes, conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol. Virulome analysis showed the presence of genes involved in adhesion and biofilm production, type II and VI secretion systems, exotoxins, etc. Multi-Locus Sequence Typing analysis identified the isolate as Sequence Types 113Pas and 2246Oxf, belonging to International Clone 7. Sequencing experiments revealed the presence of four plasmids of 2.7, 22.3, 70.4 and 240.8 Kb. All the ß-lactamase genes were located in the chromosome, except the blaPER-7, gene which was found within the plasmid of 240.8 Kb. This study highlights the threat of the emergence and dissemination of these types of isolates.

Activity of Ceftazidime-Avibactam against Clinical and Isogenic Laboratory Pseudomonas aeruginosa Isolates Expressing Combinations of Most Relevant ß-Lactam Resistance Mechanisms.

The activity of ceftazidime-avibactam was compared with that of ceftazidime alone and meropenem against a collection of 190 Pseudomonas aeruginosa clinical isolates recovered from a multicenter study of bloodstream infections. The addition of avibactam increased ceftazidime susceptibility in the complete collection of strains (64.7% to 91.1%) and particularly among subsets of isolates showing AmpC hyperproduction (10.9% to 76.1%) or multidrug resistance (MDR) profiles (27% to 77.8%). The MICs of ceftazidime-avibactam, in contrast with those of ceftazidime or meropenem, remained at ≤4 µg/ml for a panel of 16 P. aeruginosa PAO1 isogenic mutants expressing multiple combinations of the most relevant ß-lactam resistance mechanisms.

Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing.

Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones.

Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections.

BACKGROUND: The type III secretion system (TTSS) is a major virulence determinant of Pseudomonas aeruginosa. The objective of this study was to determine whether the TTSS genotype is a useful prognostic marker of P. aeruginosa bacteremia mortality. We also studied the potential association between TTSS genotypes and multidrug-resistant (MDR) profiles, and how this interaction impacts the outcome of bloodstream infections. METHODS: We performed a post hoc analysis of a published prospective multicenter cohort of P. aeruginosa bloodstream infections. The impact in mortality of TTSS genotypes (exoS, exoT, exoU, and exoY genes) and resistance profiles was investigated. Cox regression analysis was used to control for confounding variables. RESULTS: Among 590 patients, the 30-day mortality rate was 30% (175 patients), and 53% of them died in the first 5 days (early mortality). The unadjusted probabilities of survival until 5 days was 31.4% (95% confidence interval [CI], 17.4%-49.4%) for the patients with exoU-positive isolates and 53.2% (95% CI, 44.6%-61.5%) for exoU-negative isolates (log rank P = .005). After adjustment for confounders, exoU genotype (adjusted hazard ratio [aHR], 1.90 [95% CI, 1.15-3.14]; P = .01) showed association with early mortality. In contrast, late (30-day) mortality was not influenced by TTSS genotype but was independently associated with MDR profiles (aHR,1.40 [95% CI, 1.01-1.94]; P = .04). Moreover, the exoU genotype (21% of all isolates) was significantly less frequent (13%) among MDR strains (particularly among extensively drug-resistant isolates, 5%), but was positively linked to moderately resistant (1-2 antipseudomonals) phenotypes (34%). CONCLUSIONS: Our results indicate that the exoU genotype, which is associated with specific susceptibility profiles, is a relevant independent marker of early mortality in P. aeruginosa bacteremia.

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.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil.

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosa isolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding ß-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosa isolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and ß-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Within-host transition to GES-55 during a GES-6-producing Serratia marcescens outbreak: Emergence of ceftazidime-avibactam resistance and increased susceptibility to carbapenems.

OBJECTIVES: To describe the in vivo emergence of ceftazidime-avibactam resistance in GES-type carbapenemases and to characterize an unusual outbreak of GES-6-producing Serratia marcescens during the COVID-19 pandemic in Spain. METHODS: Retrospective study to describe a GES-CPSM outbreak based on whole genome sequencing and antimicrobial susceptibility testing (AST). Transferability of blaGES-carrying plasmid was assessed by conjugation experiments. RESULTS: In December 2020, we identified a cluster of S. marcescens harbouring blaGES-6 involving 9 patients. Whole-genome sequence analysis revealed a clonal relationship (≤3 SNPs) between the first isolates identified in each of the evolved patients and environmental samples with GES-CPSM detection. Plasmid analysis showed that the blaGES-6 gene was located in an IncQ3-type plasmid. Triparental mating experiments using a helper plasmid demonstrated mobilization of the blaGES-6-carrying plasmid. Our results also demonstrate within-host evolution in S. marcescens isolates, leading to a transition from blaGES-6 to the new blaGES-55, caused by the P162S mutation, in a subsequent infection in one of the affected patients. In blaGES-55 we identified emergence of ceftazidime-avibactam resistance along with an increase of carbapenems susceptibility. This patient had been treated with a 14-day course of ceftazidime-avibactam. AST of the transformants bearing blaGES-6 and blaGES-55 plasmids, confirmed susceptibility variation affecting ceftazidime-avibactam and carbapenems. CONCLUSIONS: We report an unusual outbreak of GES-6 whose incidence is becoming increasing. Transition from GES-6 to GES-55 may readily occur in vivo leading to ceftazidime-avibactam resistance, which brings to the fore the critical need for developing more accurate diagnosis tools for detection of GES ß-lactamases and optimise the use of antimicrobials.

Identification of hypermucoviscous Klebsiella pneumoniae K1, K2, K54 and K57 capsular serotypes by Raman spectroscopy.

Antimicrobial resistance poses a significant challenge in modern medicine, affecting public health. Klebsiella pneumoniae infections compound this issue due to their broad range of infections and the emergence of multiple antibiotic resistance mechanisms. Efficient detection of its capsular serotypes is crucial for immediate patient treatment, epidemiological tracking and outbreak containment. Current methods have limitations that can delay interventions and increase the risk of morbidity and mortality. Raman spectroscopy is a promising alternative to identify capsular serotypes in hypermucoviscous K. pneumoniae isolates. It provides rapid and in situ measurements with minimal sample preparation. Moreover, its combination with machine learning tools demonstrates high accuracy and reproducibility. This study analyzed the viability of combining Raman spectroscopy with one-dimensional convolutional neural networks (1-D CNN) to classify four capsular serotypes of hypermucoviscous K. pneumoniae: K1, K2, K54 and K57. Our approach involved identifying the most relevant Raman features for classification to prevent overfitting in the training models. Simplifying the dataset to essential information maintains accuracy and reduces computational costs and training time. Capsular serotypes were classified with 96 % accuracy using less than 30 Raman features out of 2400 contained in each spectrum. To validate our methodology, we expanded the dataset to include both hypermucoviscous and non-mucoid isolates and distinguished between them. This resulted in an accuracy rate of 94 %. The results obtained have significant potential for practical healthcare applications, especially for enabling the prompt prescription of the appropriate antibiotic treatment against infections.

Effect of adequate single-drug vs combination antimicrobial therapy on mortality in Pseudomonas aeruginosa bloodstream infections: a post Hoc analysis of a prospective cohort.

BACKGROUND: Empirical combination therapy is recommended for patients with known or suspected Pseudomonas aeruginosa (PA) infection as a means to decrease the likelihood of administering inadequate antimicrobial treatment, to prevent the emergence of resistance, and to achieve a possible additive or even synergistic effect. METHODS: We performed a post hoc analysis of patients with PA bloodstream infections from a published prospective cohort. Mortality was compared in patients treated with adequate empirical and definitive combination therapy (AECT, ADCT), and adequate empirical and definitive single-drug therapy (AESD, ADSD). Confounding was controlled by Cox regression analysis, and a propensity score for receiving AECT or ADCT was also used. RESULTS: The final cohort comprised 593 patients with a single episode of PA bacteremia. The 30-day mortality was 30% (176 patients); 76 patients (13%) died during the first 48 hours. The unadjusted probabilities of survival until day 30 were 69.4% (95% confidence interval [CI], 59.1-81.6) for the patients receiving AECT, 73.5% (95% CI, 68.4%-79.0%) for the AESD group, and 66.7% (95% CI, 61.2%-72.7%) for patients who received inadequate empirical therapy (P = .17, log-rank test). After adjustment for confounders, the AESD group (adjusted hazard ratio [AHR], 1.17; 95% CI, .70-1.96; P = .54) and patients who received ADSD (AHR, 1.34; 95% CI, .73-2.47; P = .35) showed no association with 30-day mortality compared with the AECT and ADCT groups, respectively. CONCLUSIONS: These results suggests that treatment with combination antimicrobial therapy did not reduce the mortality risk compared with single-drug therapy in PA bloodstream infections.

Biological markers of Pseudomonas aeruginosa epidemic high-risk clones.

A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies.

Molecular characterization of multidrug resistant Acinetobacter baumannii clinical isolates from Alexandria, Egypt.

Carbapenem resistant Acinetobacter baumannii is a major global concern, especially in countries of the Middle East and North Africa, where the antibiotic resistance rates are on the rise. The aim of this study was to study the genomic characteristics and antimicrobial susceptibility profile of thirty-six multidrug resistant A. baumannii clinical isolates obtained in hospitals from Alexandria, Egypt. Antibiotic resistance rates were estimated by determination of Minimum Inhibitory Concentrations. Carbapenemase genes, other antibiotic resistance genes and virulence factors were then screened by the use of Whole Genome Sequencing. Isolates were also subjected to Multi Locus Sequence Typing (MLST) using the Pasteur Scheme and to core genome MLST to study their clonal relatedness. In addition, plasmid analysis was performed by the use of a commercial kit and S1- Pulsed Field Gel Electrophoresis, and Hybridization experiments with DIG-labeled DNA probes for bla NDM-1, blaPER-7 and bla GES-like were performed to locate these genes. The majority of isolates were resistant to ß-lactams (including carbapenems), fluoroquinolones, aminoglycosides and trimethoprim; and some showed resistance to cefiderocol and minocycline. We identified 8 different bla OXA-51-like variants including bla OXA-51, bla OXA-64, bla OXA-65, bla OXA-66, bla OXA-68, bla OXA-91, bla OXA-94 and bla OXA-336; bla OXA-23, bla NDM-1, bla PER-7, bla GES-like and bla ADC-like and other antibiotic resistance genes, some of these genes were within transposons or class 1 integrons. Multiple virulence factors responsible for adherence, biofilm production, type II and type VI secretion systems, exotoxins, exoenzymes, immune modulation and iron uptake were observed and 34 out of 36 isolates showed motility. Thirty-five out of 36 isolates clustered with International Clones 2, 4, 5, 7, 8 and 9; and 9 STs were identified including ST570, ST2, ST600, ST15, ST113, ST613, ST85, ST158, ST164. Plasmids ranging in size from 1.7 to 70 kb were found; bla NDM-1 and blaPER-7 genes were located in the chromosome and bla GES-like genes were simultaneously located in the chromosome and in a plasmid of 70kb. In conclusion, this study revealed a wide spectrum of antibiotic resistance genes and a variety of lineages among A. baumannii isolated in hospitals from Alexandria, and highlights the importance of investigating the molecular epidemiology to control the spread of multi-drug resistant isolates.

Plasmid content of carbapenem resistant Acinetobacter baumannii isolates belonging to five International Clones collected from hospitals of Alexandria, Egypt.

Multidrug resistant Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. During the last decades it has become a major threat for healthcare settings due to the high antibiotic resistance rates among these isolates. Many resistance determinants are coded by conjugative or mobilizable plasmids, facilitating their dissemination. The majority of plasmids harbored by Acinetobacter species are less than 20 Kb, however, high molecular weight elements are the most clinically relevant since they usually contain antibiotic resistance genes. The aim of this work was to describe, classify and determine the genetic content of plasmids harbored by carbapem resistant A. baumannii isolates belonging to predominant clonal lineages circulating in hospitals from Alexandria, Egypt. The isolates were subjected to S1-Pulsed Field Gel Electrophoresis experiments to identify high molecular weight plasmids. To further analyze the plasmid content and the genetic localization of the antibiotic resistance genes, isolates were sequenced by Illumina Miseq and MinION Mk1C and a hybrid assembly was performed using Unicycler v0.5.0. Plasmids were detected with MOBsuite 3.0.3 and Copla.py v.1.0 and mapped using the online software Proksee.ca. Replicase genes were further analyzed through a BLAST against the Acinetobacter Plasmid Typing database. Eleven plasmids ranging in size from 4.9 to 205.6 Kb were characterized and mapped. All isolates contained plasmids, and, in many cases, more than two elements were identified. Antimicrobial resistance genes such as bla OXA-23, bla GES-like, aph(3')-VI and qacEΔ1 were found in likely conjugative large plasmids; while virulence determinants such as septicolysin or TonB-dependent receptors were identified in plasmids of small size. Some of these resistance determinants were, in turn, located within transposons and class 1 integrons. Among the identified plasmids, the majority encoded proteins belonging to the Rep_3 family, but replicases of the RepPriCT_1 (Aci6) family were also identified. Plasmids are of high interest as antibiotic resistance control tools, since they may be used as genetic markers for antibiotic resistance and virulence, and also as targets for the development of compounds that can inhibit transfer processes.

Automatic classification of Candida species using Raman spectroscopy and machine learning.

One of the problems that most affect hospitals is infections by pathogenic microorganisms. Rapid identification and adequate, timely treatment can avoid fatal consequences and the development of antibiotic resistance, so it is crucial to use fast, reliable, and not too laborious techniques to obtain quick results. Raman spectroscopy has proven to be a powerful tool for molecular analysis, meeting these requirements better than traditional techniques. In this work, we have used Raman spectroscopy combined with machine learning algorithms to explore the automatic identification of eleven species of the genus Candida, the most common cause of fungal infections worldwide. The Raman spectra were obtained from more than 220 different measurements of dried drops from pure cultures of each Candida species using a Raman Confocal Microscope with a 532 nm laser excitation source. After developing a spectral preprocessing methodology, a study of the quality and variability of the measured spectra at the isolate and species level, and the spectral features contributing to inter-class variations, showed the potential to discriminate between those pathogenic yeasts. Several machine learning and deep learning algorithms were trained using hyperparameter optimization techniques to find the best possible classifier for this spectral data, in terms of accuracy and lowest possible overfitting. We found that a one-dimensional Convolutional Neural Network (1-D CNN) could achieve above 80 % overall accuracy for the eleven classes spectral dataset, with good generalization capabilities.

Biochemical and genetic characterization of carbapenem-hydrolyzing ß-lactamase OXA-229 from Acinetobacter bereziniae.

Acinetobacter bereziniae (formerly Acinetobacter genomospecies 10) isolate Nec was recovered from a skin sample of a patient hospitalized in Paris, France. It was resistant to penicillins, penicillin-inhibitor combinations, and carbapenems. Cloning and expression in Escherichia coli identified the carbapenem-hydrolyzing class D ß-lactamase OXA-229, which is weakly related to other oxacillinases (66% amino acid identity with the closest oxacillinase, OXA-58). It hydrolyzed penicillins, oxacillin, and imipenem but not expanded-spectrum cephalosporins. Sequencing of the genetic context of the bla(OXA-229) gene did not identify an insertion sequence but did identify mutations in the promoter sequences in comparison to the fully susceptible A. bereziniae reference strain. The overexpression of bla(OXA-229) in A. bereziniae Nec as a source of carbapenem resistance was identified by quantitative real-time PCR.

Changes in ciprofloxacin resistance levels in Enterobacter aerogenes isolates associated with variable expression of the aac(6')-Ib-cr gene.

Two closely related Enterobacter aerogenes isolates presented a new identical aac(6')-Ib-cr genetic environment, including IS26. One isolate showed lower MICs of ciprofloxacin, norfloxacin, tobramycin, and amikacin and decreased expression of aac(6')-Ib-cr, which might be related to a 12-bp deletion causing a displacement of the -10 box upstream of the aac(6')-Ib-cr gene.

Genetic markers of widespread extensively drug-resistant Pseudomonas aeruginosa high-risk clones.

Recent reports have revealed the existence of widespread extensively drug-resistant (XDR) P. aeruginosa high-risk clones in health care settings, but there is still scarce information on their specific chromosomal (mutational) and acquired resistance mechanisms. Up to 20 (10.5%) of 190 bloodstream isolates collected from 10 Spanish hospitals met the XDR criteria. A representative number (15 per group) of isolates classified as multidrug-resistant (MDR) (22.6%), resistant to 1 to 2 classes (moderately resistant [modR]) (23.7%), or susceptible to all antibiotics (multiS) (43.2%) were investigated in parallel. Multilocus sequence typing (MLST) analysis revealed that all XDR isolates belonged to sequence type 175 (ST175) (n = 19) or ST111 (n = 1), both recognized as international high-risk clones. Clonal diversity was higher among the 15 MDR isolates (4 ST175, 2 ST111, and 8 additional STs) and especially high among the 15 modR (13 different STs) and multiS (14 STs) isolates. The XDR/MDR pattern in ST111 isolates correlated with the production of VIM-2, but none of the ST175 isolates produced acquired ß-lactamases. In contrast, the analysis of resistance markers in 12 representative isolates (from 7 hospitals) of ST175 revealed that the XDR pattern was driven by the combination of AmpC hyperproduction, OprD inactivation (Q142X), 3 mutations conferring high-level fluoroquinolone resistance (GyrA T83I and D87N and ParC S87W), a G195E mutation in MexZ (involved in MexXY-OprM overexpression), and the production of a class 1 integron harboring the aadB gene (gentamicin and tobramycin resistance). Of particular interest, in nearly all the ST175 isolates, AmpC hyperproduction was driven by a novel AmpR-activating mutation (G154R), as demonstrated by complementation studies using an ampR mutant of PAO1. This work is the first to describe the specific resistance markers of widespread P. aeruginosa XDR high-risk clones producing invasive infections.

Alterations of OprD in carbapenem-intermediate and -susceptible strains of Pseudomonas aeruginosa isolated from patients with bacteremia in a Spanish multicenter study.

We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 µg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 µg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates (77%). Isolates were classified as OprD "full-length types" (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD "deficient types" (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging to OprD "deficient types" were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of only 0.06 to 4 µg/ml.