Sara Kasraian

In Vitro Activity of the Novel Antimicrobial Peptide Dendrimer G3KL against Multidrug-Resistant Acinetobacter baumannii and Pseudomonas aeruginosa

ABSTRACT The in vitro activity of the novel antimicrobial peptide dendrimer G3KL was evaluated against 32 Acinetobacter baumannii (including 10 OXA-23, 7 OXA-24, and 11 OXA-58 carbapenemase producers) and 35 Pseudomonas aeruginosa (including 18 VIM and 3 IMP carbapenemase producers) strains and compared to the activities of standard antibiotics. Overall, both species collections showed MIC50/90 values of 8/8 μg/ml and minimum bactericidal concentrations at which 50% or 90% of strains tested are killed (MBC50/90) of 8/8 μg/ml. G3KL is a promising molecule with antibacterial activity against multidrug-resistant and extensively drug-resistant A. baumannii and P. aeruginosa isolates.

A SYBR® Green-based real-time PCR method for improved detection of mcr-1-mediated colistin resistance in human stool samples

OBJECTIVES The aim of this study was to design a rapid and sensitive real-time PCR (rt-PCR) method for colistin resistance mcr-1 gene detection in human faecal samples. METHODS Stools (n=88) from 36 volunteers were analysed. To isolate mcr-1-producing Enterobacteriaceae, samples were enriched overnight in Luria-Bertani (LB) broth containing 2mg/L colistin and were then plated on selective agar plates with 4mg/L colistin. A SYBR® Green-based rt-PCR targeting mcr-1 was then designed. For method validation and to establish the limit of detection (LOD), total DNA was extracted from mcr-1-negative and mcr-1-positive Escherichia coli. rt-PCR was also performed with DNA extracted from 88 native stools and after enriching them in LB broth containing colistin. RESULTS Based on the culture approach, three unique volunteers resulted colonised with mcr-1-harboring E. coli strains. For culture isolates, rt-PCR exhibited a LOD of 10 genomic copies/reaction, with both sensitivity and specificity of 100%. Nevertheless, when testing native stools, only two of the three mcr-1-positive specimens were detected. However, after enrichment in LB broth containing colistin, the rt-PCR was strongly positive for all culture-positive samples. The average cycle threshold was 22, granting rapid and confident detection of positive specimens within 30 cycles. No false positives were observed for the remaining 85 culture-negative specimens. CONCLUSIONS A rapid rt-PCR for detection of mcr-1 from stool specimens was developed. The detection rate was increased by testing selective broth enrichments. This approach also has the advantage of concomitant isolation of mcr-1-harboring strains for further antimicrobial susceptibility and genetic testing.

Polyclonal Intestinal Colonization with Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae upon Traveling to India.

We aimed to assess the intestinal colonization dynamics by multiple extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) clones in Swiss travelers to India, a country with high prevalence of these multidrug-resistant pathogens. Fifteen healthy volunteers (HVs) colonized with ESC-R-Ent after traveling to India who provided stools before, after, and at 3- and 6-month follow-up are presented in this study. Stools were enriched in a LB broth containing 3 mg/L cefuroxime and plated in standard selective media (BLSE, ChromID ESBL, Supercarba) to detect carbapenem- and/or ESC-R-Ent. At least 5 Enterobacteriaceae colonies were analyzed for each stool provided. All strains underwent phenotypic tests (MICs in microdilution) and molecular typing to define bla genes (microarray, PCR/sequencing), clonality (MLST, rep-PCR), and plasmid content. While only three HVs were colonized before the trip, all participants had positive stools after returning, but the colonization rate decreased during the follow-up period (i.e., six HVs were still colonized at both 3 and 6 months). More importantly, polyclonal acquisition (median of 2 clones, range 1–5) was identified at return in all HVs. The majority of the Escherichia coli isolates belonged to phylogenetic groups A and B1 and to high diverse non-epidemic sequence types (STs); however, 15% of them belonged to clonal complex 10 and mainly possessed blaCTX−M−15 genes. F family plasmids were constantly found (~80%) in the recovered ESC-R-Ent. Our results indicate a possible polyclonal acquisition of the ESC-R-Ent via food-chain and/or through an environmental exposure. For some HVs, prolonged colonization in the follow-up period was observed due to clonal persistence or presence of the same plasmid replicon types in a new bacterial host. Travel medicine practitioners, clinicians, and clinical microbiologists who are facing the returning travelers and their samples for different reasons should be aware of this important phenomenon, so that better infection control measures, treatment strategies, and diagnostic tests can be adopted.

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

ABSTRACT Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 103 to 104 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

In Vivo Evolution of CMY-2 to CMY-33 β-Lactamase in Escherichia coli Sequence Type 131: Characterization of an Acquired Extended-Spectrum AmpC Conferring Resistance to Cefepime

ABSTRACT Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical Escherichia coli isolates obtained from a patient. The CMY-2-producing E. coli isolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli isolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ec and CMY-2-Ec were identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤0.5 μg/ml for CMY-33-Ec and CMY-2-Ec, respectively). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli strain DH10B, both CMYs conferred resistance to ceftazidime (≥256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). The kcat/Km or inhibitor complex inactivation (kinact)/Ki app (μM−1 s−1) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

Intestinal colonisation with extended-spectrum cephalosporin-resistant Escherichia coli in Swiss pets: molecular features, risk factors and transmission with owners

The overlap of molecular features of extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) recovered from humans and animals indicates a possible interaction between these two niches. Frequently these strains carry CTX-M1/-14/-15 and CMY-2 β-lactamases in IncF or IncI1 plasmids in different sequence types (STs), including the high-risk clones ST131, ST648 and those belonging to clonal complex 10 (CC10) [1]. In this study, we aimed to assess the role of the intestine of healthy pets in shaping the epidemiology of ESC-R-Ent, the prevalence of intestinal colonisation by these organisms, and potential lifestyle factors associated with this phenomenon. Thus, between July 2013 and May 2016, 81 pets (44 dogs and 37 cats) and their owners (n = 72) from 66 different Swiss households were enrolled in the analysis. Owners were instructed to collect stools into sterile containers. A questionnaire about their pets was also completed. Stools were enriched overnight in Luria–Bertani broth in different conditions to detect both ESC-R-Ent and colistin-resistant (COL-R) strains [2,3]. Species identification was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonics, Leipzig, Germany). Minimum inhibitory concentrations (MICs) were determined using SensititreTM GNX2F microdilution plates (Trek Diagnostic Systems, East Grinstead, UK) and were interpreted according to 2016 European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (http://www.eucast.org/clinical_breakpoints/). β-Lactamase genes (bla) were identified using the CT103XL microarray (Check-Points, Wageningen, The Netherlands) with subsequent PCR and sequencing. Clonality was assessed by multilocus sequence typing (MLST) (http://mlst.ucc.ie/mlst/dbs/Ecoli) and phylogenetic group determination [2,3]. A PCR-based replicon typing (PBRT) kit (Diatheva, Fano, PU, Italy) was used to type plasmid incompatibility groups. Statistical analysis was performed to compare lifestyle factors of colonised and non-colonised pets using GraphPad Prism v.7.0 (GraphPad Software Inc., La Jolla, CA). Continuous variables were analysed using unpaired t-test, whereas categorical variables were analysed with Fisher’s exact test. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed for categorical variables. Among the total pets analysed, 4 cats and 3 dogs (overall prevalence, 8.6%; 95% CI 4.3–16.9%) were colonisedwith ESC-R Escherichia coli, whereas COL-R Enterobacteriaceae were not detected. Compared with previous reports, these data indicate that the prevalence is steadily increasing in Switzerland, although it is still low compared with other countries [1]. Reports on cats are scarce, with 4% reported in Kenya, 21% in Tunisia and 0% in The Netherlands [1]. Eight ESC-R E. coli isolates were recovered from seven pets; one dog was co-colonised with two different strains (Table 1). None of the isolates were resistant to carbapenems, and resistance to nonβ-lactams was low with ≤25% of the isolates non-susceptible to trimethoprim/sulfamethoxazole, aminoglycosides, quinolones and tetracyclines. Interestingly, these observations are in contrast to previous Swiss and international studies where higher resistance rates were observed [1,4]. ESC-R-Ent isolates from pets produced CTX-M-type or CMY-2 β-lactamases and carried IncF and/or IncI1 plasmids (Table 1), as reported in other international studies [1,4]. The majority of isolates belonged to phylogenetic group B1 (3/8; 37.5%) that is frequently found in the gut of animals [2]. STs were highly diverse, including CC10, CC155 and CC73, which are also found in pets and wildlife in many geographic regions [1,5]. In addition, some of these STs

Gut microbiota dynamics in travelers returning from India colonized with extended-spectrum cephalosporin-resistant Enterobacteriaceae: A longitudinal study

BACKGROUND Intestinal colonization by extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESC-R-Ent) has been attributed to travel to high prevalence countries. However, the dynamics of the microbiota changes during ESC-R-Ent colonization and whether there is a particular bacterial composition which is associated with subsequent colonization is unknown. METHODS Forty healthy volunteers living in Switzerland underwent screening before and after a trip to India, and also 3, 6 and 12 months after traveling. Culture-based ESC-R-Ent screening and microbiota analysis based on 16S rRNA amplicon sequencing were performed at all time points. RESULTS Prevalence of ESC-R-Ent colonization before traveling was 10% (n = 4), whereas it increased to 76% (n = 31) after the trip. Based on bacterial diversity analyses of the gut microbiota, there were few but significant differences for colonized versus non-colonized individuals. However, an alternative, cluster based analysis revealed that individuals remained in the same cluster over time indicating that neither traveling nor ESC-R-Ent colonization significantly influences bacterial composition. Moreover, none of the found microbiota clusters were significantly associated with subsequent risk of ESC-R-Ent colonization. CONCLUSION Based on their microbiota patterns, every volunteer was at the same risk of ESC-R-Ent colonization while traveling to India. Therefore, other risk factors for ESC-R-Ent colonization are responsible for this phenomenon.

Mismatch Amplification Mutation Assay (MAMA)-Based Real-Time PCR for Rapid Detection of and Antimicrobial Resistance Determinants in Clinical Specimens.

Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens. ABSTRACT Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens. We designed a mismatch amplification mutation assay (MAMA)-based SYBR green real-time PCR targeting one N. gonorrhoeae-specific region (opa); mosaic penA alleles (Asp345 deletion [Asp345del], Gly545Ser) associated with decreased susceptibility to cephalosporins; and alterations conferring resistance to ciprofloxacin (GyrA Ser91Phe), azithromycin (23S rRNA A2059G and C2611T), and spectinomycin (16S rRNA C1192T). We applied the real-time PCR to 489 clinical specimens, of which 94 had paired culture isolates, and evaluated its performance by comparison with the performance of commercial diagnostic molecular and phenotypic tests. Our assay exhibited a sensitivity/specificity of 93%/100%, 96%/85%, 90%/91%, 100%/100%, and 100%/90% for the detection of N. gonorrhoeae directly from urethral, rectal, pharyngeal, cervical, and vaginal samples, respectively. The MAMA strategy allowed the detection of AMR mutations by comparing cycle threshold values with the results of the reference opa reaction. The method accurately predicted the phenotype of resistance to four antibiotic classes, as determined by comparison with the MIC values obtained from 94 paired cultures (sensitivity/specificity for cephalosporins, azithromycin, ciprofloxacin, and spectinomycin resistance, 100%/95%, 100%/100%, 100%/100%, and not applicable [NA]/100%, respectively, in genital specimens and NA/72%, NA/98%, 100%/97%, and NA/96%, respectively, in extragenital specimens). False-positive results, particularly for the penA Asp345del reaction, were observed predominantly in pharyngeal specimens. Our real-time PCR assay is a promising rapid method to identify N. gonorrhoeae and predict AMR directly in genital specimens, but further optimization for extragenital specimens is needed.