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.

In Vitro Activity of 3 Commercial Bacteriophage Cocktails Against Salmonella and Shigella spp. Isolates of Human Origin.

BACKGROUND: Salmonella and Shigella spp. are 2 of the most frequent and deadly enteric bacterial pathogens recorded worldwide. In developing countries Salmonella infections are responsible for many deaths annually and these mortality rates are prone to increase due to the emergence of resistance to antibiotics. In this overall scenario new alternative therapeutic approaches are needed. METHODS: For the first time, we investigated the activity of 3 commercial bacteriophage cocktails (INTESTI, Septaphage, PYO) against a collection of contemporary Salmonella spp. (n = 30) and Shigella spp. (n = 20) strains isolated in Switzerland. Phage susceptibility was determined by implementing the spot test. RESULTS: The overall susceptibility of Salmonella spp. to INTESTI and Septaphage was 87% and 77%, respectively. With regard to Shigella spp., the overall susceptibility to INTESTI and Septaphage was 95% and 55%, respectively. PYO was observed to be active against only 10% of Salmonella spp. but against 95% of Shigella spp. CONCLUSIONS: Our results seem promising, especially for the INTESTI biopreparation against Salmonella enterica infections. Nevertheless, such speculation should be supported by further in vivo studies to confirm efficacy and safety of the cocktails. We also emphasize the importance of large in vitro screening analyses aimed to assess the activity of such biopreparations against contemporary multidrug-resistant strains that are emerging worldwide.

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.

In vitro activity of three commercial bacteriophage cocktails against multidrug-resistant Escherichia coli and Proteus spp. strains of human and non-human origin.

OBJECTIVES: Bacteriophages may represent a therapeutic alternative to treat infections caused by multidrug-resistant (MDR) pathogens. However, studies analysing their activity against MDR Enterobacteriaceae are limited. METHODS: The in vitro lytic activity of three commercial bacteriophage cocktails (PYO, INTESTI and Septaphage) was evaluated against 70 Escherichia coli and 31 Proteus spp. of human and non-human origin. Isolates were characterised by phenotypic and genotypic methods and included 82 MDR strains [44 extended-spectrum-ß-lactamase (ESBL)-producers (18 CTX-M-15-like, including ST131/ST648 E. coli); 27 plasmid-mediated AmpC ß-lactamase (pAmpC)-producers (23 CMY-2-like, including ST131 E. coli); 3 ESBL+pAmpC-producers; and 8 carbapenemase-producers]. Phage susceptibility was determined by the spot test. RESULTS: E. coli susceptibility to PYO, INTESTI and Septaphage was 61%, 67% and 9%, whereas that of Proteus spp. was 29%, 39% and 19%, respectively. For the subgroup of ESBL-producing E. coli/Proteus spp., the following susceptibility rates were recorded: PYO, 57%; INTESTI, 59%; and Septaphage, 11%. With regard to pAmpC-producers, 59%, 70% and 11% were susceptible to PYO, INTESTI and Septaphage, respectively. Five of eight carbapenemase-producers and three of four colistin-resistant E. coli were susceptible to PYO and INTESTI. CONCLUSIONS: This is the first study analysing the activity of the above three cocktails against well-characterised MDR E. coli and Proteus spp. The overall narrow spectrum of activity observed could be related to the absence of specific bacteriophages targeting these contemporary MDR strains that are spreading in different settings. Therefore, bacteriophages targeting emerging MDR pathogens need to be isolated and integrated in such biopreparations.

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 bla CTX-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.

Travelers Can Import Colistin-Resistant Enterobacteriaceae, Including Those Possessing the Plasmid-Mediated mcr-1 Gene.

Stool samples from 38 travelers returning from India were screened for extended-spectrum cephalosporin- and carbapenem-resistant Enterobacteriaceae implementing standard selective plates. Twenty-six (76.3%) people were colonized with CTX-M or DHA producers, but none of the strains was colistin resistant and/or mcr-1 positive. Nevertheless, using overnight enrichment and CHROMagar Orientation plates supplemented with colistin, four people (10.5%) were found to be colonized with colistin-resistant Escherichia coli One cephalosporin-susceptible sequence type 10 (ST10) strain carried a 4,211-bp ISApl1-mcr-1-ISApl1 element in an IncHI2 plasmid backbone.

Comparison of the in-house made Carba-NP and Blue-Carba tests: Considerations for better detection of carbapenemase-producing Enterobacteriaceae.

The in-house Carba-NP and Blue-Carba tests were compared using 30 carbapenemase- and 33 non-producing Enterobacteriaceae. Tests were read by three operators. 100% sensitivity was reported for both tests, but Carba-NP was slightly more specific than Blue-Carba (98.9% vs. 91.7%). We describe potential sources of error during tests' preparation and reading.

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

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.

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

Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) ß-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 ß-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 ß-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.

Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.

Antibiotic resistance in Ureaplasma urealyticum/Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB, parC, and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum/U. parvum, whereas 21 were positive for both U. urealyticum/U. parvum and M. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum/U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

Clonality and Antimicrobial Susceptibility of Burkholderia cepacia complex Isolates Collected from Cystic Fibrosis Patients during 1998-2013 in Bern, Switzerland.

For the first time, we analyzed the clonality and susceptibility of Burkholderia cepacia complex isolates (n=55) collected during 1998-2013 from 44 Swiss cystic fibrosis (CF)-patients. B. cenocepacia (n=28) and B. multivorans (n=14) were mainly of sequence type (ST) 833 and ST874, respectively; B. contaminans isolates were of ST102. Overall, the following MIC50/90s (mg/l) were obtained: piperacillin/tazobactam (≤ 4/≥ 128), ticarcillin/clavulanate (≥ 256/≥256), ceftazidime (2/≥ 32), aztreonam (16/≥ 32), meropenem (2/8), tobramycin (8/≥ 16), minocycline (≤ 1/16), levofloxacin (≤ 0.5/≥ 16), and trimethoprim/sulfamethoxazole (≤ 0.5/4). This is the first survey providing information on the clonality of Bcc detected in Switzerland. Species identification and antimicrobial susceptibility tests should always be routinely performed to adapt more targeted therapies.

High prevalence of extended-spectrum ß-lactamase, plasmid-mediated AmpC, and carbapenemase genes in pet food.

We evaluated the pet food contained in 30 packages as a potential origin of extended-spectrum cephalosporin-resistant Gram-negative organisms and ß-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.

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

BACKGROUND: The 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. METHODS: We 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. RESULTS: The 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). CONCLUSIONS: The 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.

Emergence of extensively drug-resistant Haemophilus parainfluenzae in Switzerland.

Two homosexual men were colonized in the urethra with Haemophilus parainfluenzae nonsusceptible to ampicillin (MIC, 8 µg/ml), amoxicillin-clavulanate (MIC, 4 µg/ml), cefotaxime (MIC, 1.5 µg/ml), cefepime (MIC, 3 µg/ml), meropenem (MIC, 0.5 µg/ml), cefuroxime, azithromycin, ciprofloxacin, tetracycline, and chloramphenicol (all MICs, ≥ 32 µg/ml). Repetitive extragenic palindromic PCR (rep-PCR) showed that the strains were indistinguishable. The isolates had amino acid substitutions in PBP3, L4, GyrA, and ParC and possessed Mef(A), Tet(M), and CatS resistance mechanisms. This is the first report of extensively drug-resistant (XDR) H. parainfluenzae.