The microbiology and pathogenesis of nonfermenting Gram-negative infections.

PURPOSE OF REVIEW: This review provides an overview of most recent evidence about pathogenesis traits and virulence factors contributing to successful colonization or infection by P. aeruginosa , A. baumannii , S. maltophilia and B. cepacia complex, among the most clinically relevant nonfermenting Gram-negative bacteria (NFGNB). RECENT FINDINGS: The growing clinical importance of NFGNB as important opportunistic pathogens causing difficult-to-treat infections in a fragile patients' population in stressed by numerous studies. Identification of novel virulence factors and deciphering of their mechanisms of action have greatly furthered our understanding of NFGNB pathogenesis, revealing that each pathogen-specific armamentarium of virulence factors (adhesins, motility, capsule, biofilm, lipopolysaccharide, exotoxins, exoenzymes, secretion systems, siderophores) can be likely responsible for the difference in the pathophysiology even in the context of a similar infection site. Emerging evidence of the immunomodulatory effect of some virulence factors is also acknowledged. SUMMARY: NFGNB continue to be a serious global problem as cause of life-threatening opportunistic infections, owing to a highly heterogeneous content of virulence factors and their extensive number of intrinsic resistance mechanisms. Further efforts in development of novel effective antimicrobials and of alternative strategies targeting key virulence factors are warranted.

Up-regulation of resident chromosomal fosB gene expression: a novel mechanism of acquired fosfomycin resistance in MRSA.

OBJECTIVES: This study investigated fosfomycin susceptibility and mechanisms of resistance in a collection of 99 Staphylococcus aureus isolated from cases of hospital-acquired pneumonia, previously collected from a multicentre survey carried out in Italy. METHODS: Fosfomycin susceptibility was tested by reference agar dilution. Bioinformatic and gene expression analysis, mutant selection experiments and WGS were executed to characterize fosfomycin resistance mechanisms. RESULTS: Fosfomycin resistance rates were 0% (0 of 35) among MSSA and 22% (14 of 64) among MRSA, with no evidence of clonal expansion. Resistance mechanisms were putatively identified in 8 of the 14 resistant strains, including: (i) chromosomal mutations causing loss of function of the UhpT transporter; (ii) overexpression of the gene encoding the Tet38 efflux pump; and (iii) overexpression of a fosB gene encoding a fosfomycin-inactivating enzyme, which was found to be resident in the chromosome of several S. aureus lineages but not always associated with fosfomycin resistance. The latter mechanism, which had not been previously described and was confirmed by results of in vitro mutant selection experiments, was associated in two cases with transposition of an IS1182 element upstream of the chromosomal fosB gene, apparently providing an additional promoter. CONCLUSIONS: This study showed that some S. aureus clonal lineages carry a resident chromosomal fosB gene and can evolve to fosfomycin resistance by overexpression of this gene.

Citrobacter freundii resistant to novel ß-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

OBJECTIVES: To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel ß-lactamase inhibitor combinations (BLICs) and cefiderocol. METHODS: Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. RESULTS: Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all ß-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. CONCLUSIONS: The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.

Mortality in KPC-producing Klebsiella pneumoniae bloodstream infections: a changing landscape.

OBJECTIVES: To assess the impact of carbapenem resistance on mortality in Klebsiella pneumoniae bloodstream infection (BSI) in the era of novel ß-lactam/ß-lactamase inhibitor combinations. MATERIAL AND METHODS: Retrospective study of patients with K. pneumoniae BSI between January and August 2020 in 16 centres (CARBANEW study within the MULTI-SITA project). RESULTS: Overall, 426 patients were included: 107/426 (25%) had carbapenem-resistant K. pneumoniae (CR-Kp) BSI and 319/426 (75%) had carbapenem-susceptible K. pneumoniae (CS-Kp) BSI. Crude cumulative 30 day mortality was 33.8% and 20.7% in patients with, respectively, CR-Kp BSI and CS-Kp BSI (P = 0.027). Carbapenemase production or carbapenemase-encoding genes were detected in 84/98 tested CR-Kp isolates (85.7%), mainly KPC (78/84; 92.9%). Ceftazidime/avibactam was the most frequently used appropriate therapy for CR-Kp BSI (80/107; 74.7%). In multivariable analyses, variables showing an unfavourable association with mortality after correction for multiple testing were age-adjusted Charlson comorbidity index (HR 1.20; 95% CI 1.10-1.31, P < 0.001) and Pitt score (HR 1.33; 95% CI 1.15-1.55, P < 0.001), but not carbapenem resistance (HR 1.28, 95% CI 0.74-2.22, P = 0.410). In a propensity score-matched analysis, there was no difference in mortality between patients appropriately treated with ceftazidime/avibactam for CR-Kp BSI and patients appropriately treated with other agents (mainly meropenem monotherapy or piperacillin/tazobactam monotherapy) for CS-Kp BSI (HR 1.07; 95% CI 0.50-2.29, P = 0.866). CONCLUSIONS: Our results suggest that the increased mortality in CR-Kp BSI compared with CS-Kp BSI is not (or no longer) dependent on the type of therapy in areas where ceftazidime/avibactam-susceptible KPC-producing isolates are the most prevalent type of CR-Kp.

Pneumocystis jirovecii pneumonia in intensive care units: a multicenter study by ESGCIP and EFISG.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is an opportunistic, life-threatening disease commonly affecting immunocompromised patients. The distribution of predisposing diseases or conditions in critically ill patients admitted to intensive care unit (ICU) and subjected to diagnostic work-up for PJP has seldom been explored. MATERIALS AND METHODS: The primary objective of the study was to describe the characteristics of ICU patients subjected to diagnostic workup for PJP. The secondary objectives were: (i) to assess demographic and clinical variables associated with PJP; (ii) to assess the performance of Pneumocystis PCR on respiratory specimens and serum BDG for the diagnosis of PJP; (iii) to describe 30-day and 90-day mortality in the study population. RESULTS: Overall, 600 patients were included in the study, of whom 115 had presumptive/proven PJP (19.2%). Only 8.8% of ICU patients subjected to diagnostic workup for PJP had HIV infection, whereas hematological malignancy, solid tumor, inflammatory diseases, and solid organ transplants were present in 23.2%, 16.2%, 15.5%, and 10.0% of tested patients, respectively. In multivariable analysis, AIDS (odds ratio [OR] 3.31; 95% confidence interval [CI] 1.13-9.64, p = 0.029), non-Hodgkin lymphoma (OR 3.71; 95% CI 1.23-11.18, p = 0.020), vasculitis (OR 5.95; 95% CI 1.07-33.22, p = 0.042), metastatic solid tumor (OR 4.31; 95% CI 1.76-10.53, p = 0.001), and bilateral ground glass on CT scan (OR 2.19; 95% CI 1.01-4.78, p = 0.048) were associated with PJP, whereas an inverse association was observed for increasing lymphocyte cell count (OR 0.64; 95% CI 0.42-1.00, p = 0.049). For the diagnosis of PJP, higher positive predictive value (PPV) was observed when both respiratory Pneumocystis PCR and serum BDG were positive compared to individual assay positivity (72% for the combination vs. 63% for PCR and 39% for BDG). Cumulative 30-day mortality and 90-day mortality in patients with presumptive/proven PJP were 52% and 67%, respectively. CONCLUSION: PJP in critically ill patients admitted to ICU is nowadays most encountered in non-HIV patients. Serum BDG when used in combination with respiratory Pneumocystis PCR could help improve the certainty of PJP diagnosis.

In vivo evolution to echinocandin resistance and increasing clonal heterogeneity in Candida auris during a difficult-to-control hospital outbreak, Italy, 2019 to 2022.

A difficult-to-control outbreak of Candida auris is ongoing in a large tertiary care hospital in Liguria, Italy, where it first emerged in 2019. In a retrospective analysis, 503 cases of C. auris carriage or infection were observed between July 2019 and December 2022. Genomic surveillance identified putative cases that no longer occurred as part of one defined outbreak and the emergence of echinocandin (pandrug) resistance following independent selection of FKS1S639F and FKS1F635Y mutants upon prolonged exposure to caspofungin and/or anidulafungin.

Long-term SARS-CoV-2 Asymptomatic Carriage in an Immunocompromised Host: Clinical, Immunological, and Virological Implications.

PURPOSE: SARS-CoV-2 infection in immunocompromised hosts is challenging, and prolonged viral shedding can be a common complication in these patients. We describe the clinical, immunological, and virological course of a patient with eosinophilic granulomatosis with polyangiitis, who developed the status of long-term asymptomatic SARS-CoV-2 carrier for more than 7 months. METHODS: Over the study period, the patient underwent 20 RT-PCR tests for SARS-CoV-2 detection on nasopharyngeal swabs. In addition, viral cultures and genetic investigation of SARS-CoV-2 were performed. As for immunological assessment, serological and specific T-cell testing was provided at different time points. RESULTS: Despite the patient showing a deep drug-induced B and T adaptive immunity impairment, he did not experience COVID-19 progression to severe complications, and the infection remained asymptomatic during the follow-up period, but he was not able to achieve viral clearance for more than 7 months. The infection was finally cleared by SARS-CoV-2-specific monoclonal antibody treatment, after that remdesivir and convalescent plasma failed in this scope. The genetic investigations evidenced that the infection was sustained by multiple viral subpopulations that had apparently evolved intra-host during the infection. CONCLUSION: Our case suggests that people with highly impaired B- and T-cell adaptive immunity can prevent COVID-19 progression to severe complications, but they may not be able to clear SARS-CoV-2 infection. Immunocompromised hosts with a long-term infection may play a role in the emergence of viral variants.

Nosocomial outbreak by NDM-1-producing Klebsiella pneumoniae highly resistant to cefiderocol, Florence, Italy, August 2021 to June 2022.

A nosocomial outbreak by cefiderocol (FDC)-resistant NDM-1-producing Klebsiella pneumoniae (NDM-Kp) occurred in a large tertiary care hospital from August 2021-June 2022 in Florence, Italy, an area where NDM-Kp strains have become endemic. Retrospective analysis of NDM-Kp from cases observed in January 2021-June 2022 revealed that 21/52 were FDC-resistant. The outbreak was mostly sustained by clonal expansion of a mutant with inactivated cirA siderophore receptor gene, which exhibited high-level resistance to FDC (MIC ≥ 32 mg/L) and spread independently of FDC exposure.

The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

BACKGROUND: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates. METHODS: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome. RESULTS: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395. CONCLUSIONS: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.

Supplementation with Lactiplantibacillus plantarum IMC 510 Modifies Microbiota Composition and Prevents Body Weight Gain Induced by Cafeteria Diet in Rats.

Changes in functionality and composition of gut microbiota (GM) have been associated and may contribute to the development and maintenance of obesity and related diseases. The aim of our study was to investigate for the first time the impact of Lactiplantibacillus (L.) plantarum IMC 510 in a rat model of diet-induced obesity, specifically in the cafeteria (CAF) diet. This diet provides a strong motivation to voluntary overeat, due to the palatability and variety of selected energy-dense foods. The oral administration for 84 days of this probiotic strain, added to the CAF diet, decreased food intake and body weight gain. Accordingly, it ameliorated body mass index, liver and white adipose tissue weight, hepatic lipid accumulation, adipocyte size, serum parameters, including glycemia and low-density lipoprotein levels, in CAF fed rats, potentially through leptin control. In this scenario, L. plantarum IMC 510 showed also beneficial effects on GM, limiting the microbial imbalance established by long exposure to CAF diet and preserving the proportion of different bacterial taxa. Further research is necessary to better elucidate the relationship between GM and overweight and then the mechanism of action by which L. plantarum IMC 510 modifies weight. However, these promising results prompt a clear advantage of probiotic supplementation and identify a new potential probiotic as a novel and safe therapeutic approach in obesity prevention and management.

Ceftazidime-Avibactam Resistance Associated with Increased blaKPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae.

This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the blaKPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.

KPC-53, a KPC-3 Variant of Clinical Origin Associated with Reduced Susceptibility to Ceftazidime-Avibactam.

This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of blaKPC-53, located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.

Results of the Italian infection-Carbapenem Resistance Evaluation Surveillance Trial (iCREST-IT): activity of ceftazidime/avibactam against Enterobacterales isolated from urine.

OBJECTIVES: To assess the in vitro antibacterial activity of ceftazidime/avibactam against a recent Italian collection of carbapenem-resistant Enterobacterales (CRE) isolated from urine specimens. METHODS: Consecutive Gram-negative isolates from urine specimens, collected from inpatients in five Italian hospitals during the period October 2016 to February 2017, were screened for CRE phenotype using chromogenic selective medium and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed by reference broth microdilution (BMD) and, for ceftazidime/avibactam, also by Etest® CZA. Results were interpreted according to the EUCAST breakpoints. All confirmed CRE were subjected to real-time PCR targeting blaKPC-type, blaVIM-type, blaNDM-type and blaOXA-48-type carbapenemase genes. Non-MBL-producing isolates resistant to ceftazidime/avibactam were subjected to WGS and their resistome and clonality were analysed. RESULTS: Overall, 318 non-replicate presumptive CRE were collected following screening of 9405 isolates of Enterobacterales (3.4%) on chromogenic selective medium. Molecular analysis revealed that 216 isolates were positive for a carbapenemase gene (of which 92.1%, 2.8%, 1.4% and 1.4% were positive for blaKPC-type, blaOXA-48-type, blaNDM-type and blaVIM-type, respectively). Against the confirmed carbapenemase-producing Enterobacterales (CPE), ceftazidime/avibactam was the most active compound, followed by colistin (susceptibility rates 91.6% and 69.4%, respectively). Compared with BMD, Etest® for ceftazidime/avibactam yielded consistent results (100% category agreement). All class B ß-lactamase producers were resistant to ceftazidime/avibactam, while OXA-48 and KPC producers were susceptible, with the exception of seven KPC-producing isolates (4.2%). The latter exhibited an MIC of 16 to >32 mg/L, belonged to ST512, produced KPC-3 and showed alterations in the OmpK35 and Ompk36 porins. CONCLUSIONS: Ceftazidime/avibactam showed potent in vitro activity against a recent Italian collection of CPE from urine.

Influence of a 3-month low-calorie Mediterranean diet compared to the vegetarian diet on human gut microbiota and SCFA: the CARDIVEG Study.

PURPOSE: We evaluated the effect of low-calorie mediterranean (MD) and vegetarian (VD) diets on gut microbiome (GM) composition and short-chain-fatty acids (SCFA) production. METHODS: We performed next generation sequencing (NGS) of 16S rRNA and SCFA analysis on fecal samples of 23 overweight omnivores (16 F; 7 M) with low-to-moderate cardiovascular risk. They were randomly assigned to a VD or MD, each lasting 3 months, with a crossover study design. RESULTS: Dietary interventions did not produce significant diversity in the GM composition at higher ranks (family and above), neither between nor within MD and VD, but they did it at genus level. MD significantly changed the abundance of Enterorhabdus, Lachnoclostridium and Parabacteroides, while VD significantly affected the abundance of Anaerostipes, Streptococcus, Clostridium sensu stricto, and Odoribacter. Comparison of the mean variation of each SCFA between MD and VD showed an opposite and statistically significant trend for propionic acid (+ 10% vs - 28%, respectively, p = 0.034). In addition, variations of SCFA were negatively correlated with changes of some inflammatory cytokines such as VEGF, MCP-1, IL-17, IP-10 and IL-12, only after MD. Finally, correlation analyses showed a potential relationship-modulated by the two diets-between changes of genera and changes of clinical and biochemical parameters. CONCLUSIONS: A short-term dietary intervention with MD or VD does not induce major change in the GM, suggesting that a diet should last longer than 3 months for scratching the microbial resilience. Changes in SCFA production support their role in modulating the inflammatory response, thus mediating the anti-inflammatory and protective properties of MD.

Characterization of Tn6349, a novel mosaic transposon carrying poxtA, cfr and other resistance determinants, inserted in the chromosome of an ST5-MRSA-II strain of clinical origin.

OBJECTIVES: To characterize the genetic element carrying the poxtA oxazolidinone resistance gene found in the poxtA index strain Staphylococcus aureus AOUC-0915 isolated from a cystic fibrosis patient. METHODS: The genetic context of poxtA was investigated by bioinformatics analysis of WGS data of strain AOUC-0915, followed by PCR and confirmatory Sanger sequencing for repetitive regions. Conjugation and electrotransformation experiments were carried out to assess horizontal transferability using S. aureus and Enterococcus faecalis recipients. Production of phage particles was evaluated by PCR using DNA preparations obtained after phage induction. Excision of the transposon carrying poxtA was evaluated by inverse PCR experiments for detection of circular intermediates. RESULTS: poxtA was found to be associated with a 48 kb composite transposon of original structure, named Tn6349, inserted into a φN315-like prophage. The transposon was bounded by two IS1216 insertion sequences, carried several resistance genes [erm(B), cfr, poxtA and fexB] and exhibited a mosaic structure made by a derivative of plasmid pE35048-oc (previously described in an Enterococcus faecium clinical isolate) and Tn6657, a novel composite transposon carrying the poxtA and fexB genes. Excision ability of Tn6349 as a circular intermediate was demonstrated. Transferability of Tn6349 or modules thereof to S. aureus or E. faecalis by either conjugation or electrotransformation was not detected. Induction of the φN315-like prophage carrying Tn6349 was not observed. CONCLUSIONS: This study describes the structure of Tn6349, a novel composite transposon carrying several resistance determinants to anti-ribosomal drugs, including cfr and poxtA, from an oxazolidinone-resistant MRSA strain. Analysis of Tn6349 revealed a modular structure that could favour the mobilization of its resistance determinants.

Staphylococcus aureus from hospital-acquired pneumonia from an Italian nationwide survey: activity of ceftobiprole and other anti-staphylococcal agents, and molecular epidemiology of methicillin-resistant isolates.

OBJECTIVES: To determine the prevalence of Staphylococcus aureus from hospital-acquired pneumonia (HAP) in Italy and the susceptibility to ceftobiprole and comparators of MSSA and MRSA isolates. A secondary objective was to characterize the clonality and acquired resistance and virulence genes of MRSA. METHODS: Consecutive non-replicate isolates from HAP were collected from 13 laboratories distributed across Italy, from January to May 2016. Antimicrobial susceptibility testing was performed by broth microdilution, and results were interpreted according to the EUCAST breakpoints. All MRSA isolates were subjected to WGS using an Illumina platform. Clonality and resistance and virulence gene content were investigated with bioinformatics tools. RESULTS: Among 333 isolates from HAP, S. aureus was the third most common pathogen (18.6%). The proportion of MRSA was 40.3%. Susceptibility to ceftobiprole was 100% for MSSA and 95.5% for MRSA. Lower susceptibility rates of 78.4% and 94.6% in MSSA and 36.4% and 12.1% in MRSA isolates were observed for erythromycin and levofloxacin, respectively. The MRSA from HAP mostly belonged to clonal complex (CC) 22 (47.0%), CC5 (25.8%) and CC8 (15.2%), with a minority of other lineages (ST1, ST6, ST7, ST30, ST152 and ST398). Acquired resistance and virulence genes in most cases exhibited a clonal distribution. The three ceftobiprole-resistant isolates exhibited an MIC of 4 mg/L and belonged to ST228-MRSA-I of CC5. CONCLUSIONS: S. aureus is an important cause of HAP in Italy. Ceftobiprole exhibited good in vitro activity against S. aureus isolated from HAP, including MRSA. A trend to replacement of ST228 with ST22 was noticed compared with previous studies.

Characterization of the first blaCTX-M-14/ermB-carrying IncI1 plasmid from Latin America.

Azithromycin represents a valid therapeutic option for gastrointestinal and systemic infections caused by multidrug resistant (MDR) diarrheagenic Escherichia coli, Shigella and Salmonella species. However, acquired macrolide resistance in these species has been increasingly described, mostly related to macrolide modifying enzymes encoded by mph- and erm-type genes. In this study, we characterized the first blaCTX-M-14/ermB-carrying IncI1 plasmid from Latin America, which was detected in a MDR E. coli clinical isolate from Uruguay. Plasmid pUR-EC07 was a 105,836-bp IncI1 ST80 plasmid harboring blaCTX-M-14,blaTEM-1 and ermB, and characterized by a backbone region of 92,554 bp, and a multi resistance region (MRR) of 13,282 bp, bounded by two directly-oriented IS26 elements, and inserted upstream of ResD. The MRR comprised two nested IS26-composite transposons (Tn6651 and Tn6652). Tn6651 included the ermBL-ermB operon flanked by two IS26 copies in opposite direction. Tn6652 included blaTEM-1, with an upstream remnant of a Tn3-family transposon, and blaCTX-M-14, located in a genetic context consisting of ISEcp1∆-IS10-blaCTX-M-14-IS903D. To the best of our knowledge, pUR-EC07 represents the first IncI1 plasmid harboring ermB and blaCTX-M-14 described in Latin America. Given the wide dissemination of IncI-type plasmids in Salmonella enterica, an important concern is represented by the potential transfer of similar plasmids to strains resistant to ciprofloxacin, which would result in the loss of the three main therapeutic resources.

Characterization of Extensively Drug-Resistant or Pandrug-Resistant Sequence Type 147 and 101 OXA-48-Producing Klebsiella pneumoniae Causing Bloodstream Infections in Patients in an Intensive Care Unit.

Carbapenem-resistant Klebsiella pneumoniae causes important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae (OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB (distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.

Italian nationwide survey on Pseudomonas aeruginosa from invasive infections: activity of ceftolozane/tazobactam and comparators, and molecular epidemiology of carbapenemase producers.

Objectives: Pseudomonas aeruginosa is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Ceftolozane/tazobactam is a new cephalosporin/ß-lactamase inhibitor combination with potent activity against P. aeruginosa. This survey was carried out to evaluate the susceptibility of P. aeruginosa, circulating in Italy, to ceftolozane/tazobactam and comparators and to investigate the molecular epidemiology of carbapenemase-producing strains. Methods: Consecutive non-replicate P. aeruginosa clinical isolates (935) from bloodstream infections and lower respiratory tract infections were collected from 20 centres distributed across Italy from September 2013 to November 2014. Antimicrobial susceptibility testing was performed by broth microdilution and results were interpreted according to the EUCAST breakpoints. Isolates resistant to ceftolozane/tazobactam were investigated for carbapenemase genes by PCR, and for carbapenemase activity by spectrophotometric assay. WGS using an Illumina platform was performed on carbapenemase-producing isolates. Results: Ceftolozane/tazobactam was the most active molecule, retaining activity against 90.9% of P. aeruginosa isolates, followed by amikacin (88.0% susceptibility) and colistin (84.7% susceptibility). Overall, 48 isolates (5.1%) were positive for carbapenemase genes, including blaVIM (n = 32), blaIMP (n = 12) and blaGES-5 (n = 4), while the remaining ceftolozane/tazobactam-resistant isolates tested negative for carbapenemase production. Carbapenemase producers belonged to 10 different STs, with ST175 (n = 12) and ST621 (n = 11) being the most common lineages. Genome analysis revealed different trajectories of spread for the different carbapenemase genes. Conclusions: Ceftolozane/tazobactam exhibited potent in vitro activity against P. aeruginosa causing invasive infections in Italy. Carbapenemase production was the most common mechanism of resistance to ceftolozane/tazobactam.

In vitro synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii grown in planktonic phase and in biofilms.

Objectives: To investigate the potential synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii strains grown in planktonic phase or as biofilms. Methods: Sixteen strains were investigated, including nine colistin-susceptible (MIC range 0.5-1 mg/L) and seven colistin-resistant (MIC range 16-256 mg/L) strains. Synergism of colistin in combination with N-acetylcysteine was investigated by chequerboard assays. The activity of colistin/N-acetylcysteine combinations was further evaluated by time-kill assays with planktonic cultures (three colistin-resistant strains and one colistin-susceptible strain) and by in vitro biofilm models (three colistin-resistant and three colistin-susceptible strains). Results: Chequerboard assays revealed a relevant synergism of colistin/N-acetylcysteine combinations with all colistin-resistant strains, whereas no synergism was observed with colistin-susceptible strains. Time-kill assays showed a concentration-dependent potentiation of colistin activity by N-acetylcysteine against colistin-resistant strains, with eradication of the culture by combinations of N-acetylcysteine at 8000 mg/L plus colistin at 2 or 8 mg/L. A static effect during the first 8 h of incubation was demonstrated with the colistin-susceptible strain exposed to 0.25 × MIC colistin plus 8000 mg/L N-acetylcysteine. A remarkable antibiofilm synergistic activity of 8 mg/L colistin plus 8000 mg/L N-acetylcysteine was demonstrated with all colistin-resistant and colistin-susceptible strains. The effects were greater with colistin-resistant strains (marked reduction of viable biofilm cells was observed at sub-MIC colistin concentrations). Conclusions: N-acetylcysteine, at concentrations achievable by topical administration, was shown to revert the colistin-resistant phenotype in A. baumannii, and to exert a relevant activity against biofilms of colistin-susceptible and colistin-resistant A. baumannii strains.