Agnese Lupo

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins

BackgroundThe spread of Neisseria gonorrhoeae (Ng) isolates resistant to the clinically implemented antibiotics is challenging the efficacy of treatments. Unfortunately, phenotypic and molecular data regarding Ng detected in Switzerland are scarce.MethodsWe compared the characteristics of Ng detected during 1998–2001 (n = 26) to those detected during 2009–2012 (n = 34). MICs were obtained with the Etest and interpreted as non-susceptible (non-S) according to EUCAST criteria. Sequence type (ST) was achieved implementing the NG-MAST. BlaTEM, ponA, penA, mtrR, penB, tet(M), gyrA, parC, mefA, ermA/B/C/F, rplD, rplV, and 23S rRNA genes were analyzed.ResultsThe following susceptibility results were obtained (period: % of non-S, MIC90 in mg/L): penicillin (1998–2001: 42.3%, 3; 2009–2012: 85.3%, 16), cefixime (1998–2001: 0%, ≤0.016; 2009–2012: 8.8%, 0.125), ceftriaxone (1998–2001: 0%, 0.004; 2009–2012: 0%, 0.047), ciprofloxacin (1998–2001: 7.7%, 0.006; 2009–2012: 73.5%, ≥32), azithromycin (1998–2001: 11.5%, 0.25; 2009–2012: 23.6%, 0.38), tetracycline (1998–2001: 65.4%, 12; 2009–2012: 88.2%, 24), spectinomycin (1998–2001: 0%, 12; 2009–2012: 0%, 8). The prevalence of multidrug-resistant (MDR) isolates increased from 7.7% in 1998–2001 to 70.6% in 2009–2012. International STs and genogroups (G) emerged during 2009–2012 (G1407, 29.4%; G2992, 11.7%; G225, 8.8%). These isolates possessed distinctive mechanisms of resistance (e.g., G1407: PBP1 with L421, PBP2 pattern XXXIV, GyrA with S91F and D95G, ParC with S87R, PorB with G120K and A121N, mtrR promoter with A deletion).ConclusionsThe prevalence of penicillin- ciprofloxacin- and tetracycline-resistant Ng has reached dramatic levels, whereas cefixime and ceftriaxone show MICs that tend to increase during time. International MDR clones less susceptible to cephalosporins are rapidly emerging indicating that the era of untreatable gonococcal infections is close.

High Prevalence of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae in Poultry Meat in Switzerland: Emergence of CMY-2- and VEB-6-possessing Proteus mirabilis

The spread of extended-spectrum-cephalosporin-resistant (ESC-R) Escherichia coli in poultry meat is a serious concern ([1][1][–][2][3][3]). However, data regarding this problem in Switzerland are lacking. Moreover, the role played in this matter by other Enterobacteriaceae remains undetermined.

Antibiotic resistance and phylogenetic characterization of Acinetobacter baumannii strains isolated from commercial raw meat in Switzerland.

The spread of antibiotic-resistant bacteria through food has become a major public health concern because some important human pathogens may be transferred via the food chain. Acinetobacter baumannii is one of the most life-threatening gram-negative pathogens; multidrug-resistant (MDR) clones of A. baumannii are spreading worldwide, causing outbreaks in hospitals. However, the role of raw meat as a reservoir of A. baumannii remains unexplored. In this study, we describe for the first time the antibiotic susceptibility and fingerprint (repetitive extragenic palindromic PCR [rep-PCR] profile and sequence types [STs]) of A. baumannii strains found in raw meat retailed in Switzerland. Our results indicate that A. baumannii was present in 62 (25.0%) of 248 (CI 95%: 19.7 to 30.9%) meat samples analyzed between November 2012 and May 2013, with those derived from poultry being the most contaminated (48.0% [CI 95%: 37.8 to 58.3%]). Thirty-nine strains were further tested for antibiotic susceptibility and clonality. Strains were frequently not susceptible (intermediate and/or resistant) to third- and fourth-generation cephalosporins for human use (i.e., ceftriaxone [65%], cefotaxime [32%], ceftazidime [5%], and cefepime [2.5%]). Resistance to piperacillin-tazobactam, ciprofloxacin, colistin, and tetracycline was sporadically observed (2.5, 2.5, 5, and 5%, respectively), whereas resistance to carbapenems was not found. The strains were genetically very diverse from each other and belonged to 29 different STs, forming 12 singletons and 6 clonal complexes (CCs), of which 3 were new (CC277, CC360, and CC347). RepPCR analysis further distinguished some strains of the same ST. Moreover, some A. baumannii strains from meat belonged to the clonal complexes CC32 and CC79, similar to the MDR isolates responsible for human infections. In conclusion, our findings suggest that raw meat represents a reservoir of MDR A. baumannii and may serve as a vector for the spread of these pathogens into both community and hospital settings.

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

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.

Is Penicillin Plus Gentamicin Synergistic against Clinical Group B Streptococcus isolates?: An In vitro Study

Group B Streptococcus (GBS) is increasingly causing invasive infections in non-pregnant adults. Elderly patients and those with comorbidities are at increased risk. On the basis of previous studies focusing on neonatal infections, penicillin plus gentamicin is recommended for infective endocarditis (IE) and periprosthetic joint infections (PJI) in adults. The purpose of this study was to investigate whether a synergism with penicillin and gentamicin is present in GBS isolates that caused IE and PJI. We used 5 GBS isolates, two clinical strains and three control strains, including one displaying high-level gentamicin resistance (HLGR). The results from the checkerboard and time-kill assays (TKAs) were compared. For TKAs, antibiotic concentrations for penicillin were 0.048 and 0.2 mg/L, and for gentamicin 4 mg/L or 12.5 mg/L. In the checkerboard assay, the median fractional inhibitory concentration indices (FICIs) of all isolates indicated indifference. TKAs for all isolates failed to demonstrate synergism with penicillin 0.048 or 0.2 mg/L, irrespective of gentamicin concentrations used. Rapid killing was seen with penicillin 0.048 mg/L plus either 4 mg/L or 12.5 mg/L gentamicin, from 2 h up to 8 h hours after antibiotic exposure. TKAs with penicillin 0.2 mg/L decreased the starting inoculum below the limit of quantification within 4–6 h, irrespective of the addition of gentamicin. Fast killing was seen with penicillin 0.2 mg/L plus 12.5 mg/L gentamicin within the first 2 h. Our in vitro results indicate that the addition of gentamicin to penicillin contributes to faster killing at low penicillin concentrations, but only within the first few hours. Twenty-four hours after antibiotic exposure, PEN alone was bactericidal and synergism was not seen.

High Prevalence of Extended-Spectrum β-Lactamase, Plasmid-Mediated AmpC, and Carbapenemase Genes in Pet Food

ABSTRACT We evaluated the pet food contained in 30 packages as a potential origin of extended-spectrum cephalosporin-resistant Gram-negative organisms and β-lactamase genes (bla). Live bacteria were not detected by selective culture. However, PCR investigations on food DNA extracts indicated that samples harbored the blaCTX-M-15 (53.3%), blaCMY-4 (20%), and blaVEB-4-like (6.7%) genes. Particularly worrisome was the presence of blaOXA-48-like carbapenemases (13.3%). The original pet food ingredients and/or the production processes were highly contaminated with bacteria carrying clinically relevant acquired bla genes.

Raw meat contaminated with epidemic clones of Burkholderia multivorans found in cystic fibrosis patients

Burkholderia multivorans is a Gram-negative pathogen belonging to the Burkholderia cepacia complex (Bcc). Together with Burkholderia cenocepacia it is the most prevalent Bcc species causing respiratory tract infection or colonization in cystic fibrosis (CF)-patients. Due to the natural multidrug resistant (MDR) pattern and the ability to form biofilm, antibiotic treatment of Bcc infections is difficult and frequently results in high morbidity and mortality [1]. The implementation of the multi-locus sequence typing (MLST) for the Bcc species has highlighted that some B. multivorans sequence types (STs; i.e., ST16, ST17, ST24, ST181, ST190, ST195, ST274, ST374, and ST439) are internationally distributed among CF-patients [2,3]. In particular, ST16 is an epidemic clone responsible for outbreaks in at least six different countries (i.e., France, Belgium, Canada, United States, Australia, and New Zealand), whereas ST24 was reported in Canada and Brazil [3]. Environment, as well as materials and products that come into a hospital, may constitute sources of infection with B. cenocepacia. In contrast, it seems that the spread of B. multivorans occurs with different dynamics. In particular, B. multivorans is rarely isolated from natural environment

First report of the macrolide efflux genetic assembly (MEGA) element in Haemophilus parainfluenzae.

We previously described two identical extensively drug-resistant (XDR) Haemophilus parainfluenzae isolates non-susceptible to β-lactams, macrolides, quinolones, tetracycline and chloramphenicol [1]. In an effort to analyse how the resistance genes of these pathogens were organised, one isolate (AE-2096513) underwent Illumina HiSeq 200 whole-genome sequencing. Genomic DNA was extracted and multiplexed paired-end libraries were created using the sample preparation kit from Illumina Inc. (San Diego, CA). SPAdes v.2.5 (kmer sizes = 33, 55, 67, 81, 91, 93, 95, 97, 99) was used for read correction and de novo assembly [2]. The resulting number of scaffolds and total sequence length was 2.71 Mb (GenBank accession no. MOTP00000000). Scaffolds with a length of <500 bp were extracted, resulting in 124 scaffolds that had more than 100-fold read coverage. Special emphasis was then placed on the genetic environment of mef(E)/mel (encoding a dual efflux pump affecting macrolides), often found within the macrolide efflux genetic assembly (MEGA) element, and tet(M) (encoding a ribosomal protective protein against tetracyclines). The Illumina reads weremapped using Bowtie2 v.2.1.0 to the MEGA element (accession no. FR671415). The reads pairs, of which only one mate mapped to the MEGA element, were extracted. The non-mapping mates were aligned to the genome sequence of the reference H. parainfluenzae T3T1 (accession no. YP_004823607) using Basic Local Alignment Search Tool (BLAST) to specifically focus on the flanking regions of the tet(M)–MEGA element (accession no. KJ545575; see results below). Using this information, a fusion sequence including the tet(M)– MEGA element was constructed in silico (Fig. 1). Based on this result, sequence primers were designed to confirm the chromosomal insertion site of the element using traditional Sanger sequencing. First, using the tet(M)–MEGA flanking primers RF-end (5′GGTTTAACCGAATGGGCGCCTGC-3′) and 19210rev (5′-CTTGCAGT CAAATAGTAGTTGG-3′), a PCRproductwith theexpected insert site (ca. 10 kb) was obtained. Second, it was revealed that the tet(M)–MEGA elementwas inserted 2 bpdownstreamof RF-3 (ORF19190) at position 1 989 391 compared with H. parainfluenzae T3T1 using primers RFendandMEGA-end(5′-TTTATTTAAGAATACCTTGCCGC-3′).Third,primers mef(E)F (5′-TTCTTCTGGTACTAAAAGTGG-3′) and 19210rev were used to obtain a PCR product of 2.1 kb, which was subsequently sequenced using an additional internal primer (IMLS2, 5′-TCCCGCACCA TTTATCAGCTT-3′). This revealed that the downstream insertion site of tet(M)–MEGA in AE-2096513 is at position 1 989 935 compared with T3T1, therefore replacing ORF19200 (Fig. 1). Finally, we closely inspected the ends of the element, showing that there are no inverted repeats rendering difficult a possible explanation for the mechanism of insertion (Fig. 1). The MEGA element has been previously reported in streptococcal species such as Streptococcus salivarius and Streptococcus pneumoniae [3]. For S. salivarius, no adjacent tet(M) was described, whilst for S. pneumoniae tet(M) upstream of the MEGA element and within transposon Tn916 has been frequently identified (Fig. 1). Tn916 and the often co-identified composite transposons (e.g. Tn2009 and Tn2010) are thought of being mobile by transformation, and more than four distinct insertion sites have been identified within the pneumococcal genome [4]. In AE-2096513, tet(M) together with MEGA were in a ‘non-Tn916’ context because Tn916 typically contains the ORF8 to 23 (as illustrated for S. pneumoniae 23771) that were absent in our element (Fig. 1). In conclusion, we identified for the first time the tet(M)–MEGA element in an H. parainfluenzae isolate. This element is probably mobilised and might be transferred between different members of the microbial community. In the case of H. parainfluenzae AE2096513, this strain has been isolated along with a pan-susceptible Neisseria gonorrhoeae from a urethral swab [1]. H. parainfluenzaewas therefore considered as a coloniser, but it would be particularly worrisome if the newly described tet(M)–MEGA could be exchangedwith N. gonorrhoeae, conferring resistance to azithromycin, one of the standard therapeutic options [5]. However, H. parainfluenzae is normally isolated from the pharynx, where there are also streptococci and pneumococci. Considering the close similarity of the tet(M)– MEGA elements of H. parainfluenzae AE-2096513 and S. pneumoniae 23711, genetic exchange between these two bacterial species is likely. These findings may therefore serve as a paradigmatic example of microbial acquisition of mobile genetic elements carrying resistance genes against clinically used antibiotics.