Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates.

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients.

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.

TEM-184, a Novel TEM-Derived Extended-Spectrum ß-Lactamase with Enhanced Activity against Aztreonam.

TEM-184, a novel TEM-derived extended-spectrum ß-lactamase (ESBL), was isolated from an Escherichia coli ST354 clinical strain. Compared to TEM-1, TEM-184 contains the mutations Q6K, E104K, I127V, R164S, and M182T. Kinetic analysis of this enzyme revealed extended-spectrum activity against aztreonam in particular. TEM-184 was also susceptible to inhibitors, including clavulanic acid, tazobactam, and avibactam.

First report of NDM-1-producing Klebsiella pneumoniae imported from Africa to Italy: Evidence of the need for continuous surveillance.

OBJECTIVES: NDM-producing Enterobacteriaceae are considered emergent on the African continent and have been increasingly reported in recent years. In contrast, strains producing NDM-type enzymes have been rarely reported in Italy, usually associated with sporadic cases or small outbreaks. Here we report two cases of infection caused by NDM-1-producing Klebsiella pneumoniae (NDM-KP) in two unrelated patients returned from travel to Egypt. CASE REPORTS: The two patients had been previously hospitalised for a short period in two different Egyptian hospitals. In our institution in Italy, NDM-KP isolates were detected from surgical wound drainage (patient #1) and respiratory secretions and blood cultures (patient #2). Rectal swabs of both patients were persistently positive for NDM-KP. In both cases, NDM-1-producing isolates exhibited a multidrug-resistant phenotype, being susceptible only to tigecycline and colistin. Analysis by multilocus sequence typing (MLST) revealed that the two K. pneumoniae isolates were not clonally related, belonging to different sequence types (STs), namely ST15 from patient #1 and ST11 from patient #2. CONCLUSIONS: To the best of our knowledge, this is the first report of NDM-producing isolates imported from Africa to Italy, with no obvious link to the Indian subcontinent. Our experience confirms that Egypt is an emergent source of NDM-producing Enterobacteriaceae, thus representing a cause of concern for Mediterranean countries. Owing to its geographical position, Italy is a first-line European checkpoint with respect to African countries and plays a pivotal role in limiting the dissemination of high-risk clones, especially considering the latest strong migration flows.

Molecular epidemiology of KPC-producing Klebsiella pneumoniae from invasive infections in Italy: increasing diversity with predominance of the ST512 clade II sublineage.

OBJECTIVES: The spread of carbapenem-resistant Enterobacteriaceae (CRE) represents one of the most worrisome problems for clinical medicine worldwide. In Italy, the Antibiotic-Resistance-Istituto Superiore di Sanità surveillance network, in collaboration with the Committee for Antimicrobial Agents of the Italian Society of Clinical Microbiologists, promoted a study to investigate the carbapenem-resistance mechanisms, clonal relatedness and capsular typing of a recent collection of carbapenem-resistant Klebsiella pneumoniae (CR-KP). METHODS: A total of 17 laboratories distributed across Italy collected all consecutive non-replicate CR-KP isolated from invasive infections during two different study periods (2011-12 and 2013). Carbapenemase genes were searched for by filter hybridization and confirmed by PCR and sequencing. KPC-producing K. pneumoniae (KPC-KP) were typed by PFGE and MLST. Capsular types were identified by wzi gene typing. RESULTS: Of the collected K. pneumoniae isolates (n = 461), the overall proportion of CR-KP was 36.2% (n = 167). The majority (97%) of the CR-KP were positive for the blaKPC gene. Among the KPC-KP population, nine different STs were detected with the majority of isolates (94%) belonging to the clonal group (CG) 258. A subpopulation that belonged to ST512 and showed an identical PFGE profile represented the majority (57%) of KPC-KP strains, with a countrywide distribution. Capsular characterization showed the predominance of the wzi154, cps-2 capsular type (88.8% of all CG258 strains). ST258 strains were associated with both cps-1 and cps-2 capsular types, while ST512 was associated with cps-2 only. CONCLUSIONS: Although a trend to a polyclonal evolution of the Italian KPC-KP was noted, this study showed that the KPC-KP population remained largely oligoclonal with the wide diffusion of an ST512 lineage carrying cps-2 capsular type and producing the KPC-3 enzyme.

Large Nosocomial Outbreak of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Traced to Clonal Expansion of an mgrB Deletion Mutant.

We describe a large hospital outbreak (93 bloodstream infections) of colistin-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates which was mirrored by increased colistin consumption. The outbreak was mostly traced to the clonal expansion of an mgrB deletion mutant of an ST512 strain that produced KPC-3.

Catheter-related bacteremia by Cupriavidus metallidurans.

Cupriavidus bacteremia is a rare infection and identification of the pathogen is difficult. We present four cases of bacteremia by Cupriavidus metallidurans that were initially identified to the genus level by both Bruker and Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry and later identified to the species level by 16S rRNA gene sequencing. To our knowledge, these are the first cases of C. metallidurans catheter-related infections. Patients were successfully treated with antibiotic therapy and catheter removal.

MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin.

Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP) are challenging multidrug-resistant pathogens due to their extensively drug-resistant phenotypes and potential for epidemic dissemination in health care settings. Colistin is a key component of the combination antimicrobial regimens used for treatment of severe KPC-KP infections. We previously reported that insertional inactivation of the mgrB gene, encoding a negative-feedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-KP, due to the resulting upregulation of the Pmr lipopolysaccharide modification system. In this work we investigated the status of the mgrB gene in a collection of 66 colistin-resistant nonreplicate clinical strains of KPC-KP isolated from different hospitals in Italy and Greece. Overall, 35 strains (53%) exhibited alterations of the mgrB gene, including insertions of different types of mobile elements (IS5-like, IS1F-like, or ISKpn14), nonsilent point mutations, and small intragenic deletions. Four additional strains had a larger deletion of the mgrB locus, while the remaining 27 strains (41%) did not show mgrB alterations. Transcriptional upregulation of the phoQ and pmrK genes (part of the phoPQ and pmrHFIJKLM operon, respectively) was observed in all strains with mgrB alterations. Complementation experiments with a wild-type mgrB gene restored colistin susceptibility and basal expression levels of phoQ and pmrK genes in strains carrying different types of mgrB alterations. The present results suggest that mgrB alteration can be a common mechanism of colistin resistance among KPC-KP in the clinical setting.

Characterization of pFOX-7a, a conjugative IncL/M plasmid encoding the FOX-7 AmpC-type ß-lactamase, involved in a large outbreak in a neonatal intensive care unit.

OBJECTIVES: FOX-type enzymes are a lineage of AmpC-type ß-lactamases from Aeromonas spp. whose genes have been mobilized to plasmids spreading among Enterobacteriaceae, where they can be responsible for resistance to extended-spectrum cephalosporins and ß-lactamase inhibitor combinations. Little is known about the genetic context and plasmid vehicles of bla(FOX) determinants. Here, we have characterized a plasmid encoding the FOX-7 ß-lactamase, which was involved in a large outbreak caused by two Klebsiella pneumoniae clones in a neonatal intensive care unit. METHODS: Plasmid transferability was tested in conjugation experiments using Escherichia coli recipients. Plasmids from different strains were compared by restriction profiling and PCR mapping. The complete sequence of pFOX-7a plasmid was determined by a next-generation sequencing approach followed by gap filling using PCR and sequencing. RESULTS: An apparently identical conjugative plasmid encoding FOX-7 was detected in representatives of the K. pneumoniae clones that caused the outbreak and in sporadic FOX-7-producing strains of other species from the same ward. The plasmid, named pFOX-7a, has an IncL/M-type backbone and two separate resistance modules including a Tn3-like transposon and a novel Tn1696 derivative, named Tn6234, which carries an integron platform, a hybrid (but still functional) mercury resistance module and a novel putative transposon of original structure, named Tn6240, associated with the bla(FOX-7) gene. CONCLUSIONS: pFOX-7a is the first completely characterized plasmid encoding a FOX-type ß-lactamase. The bla(FOX-7) gene was associated with a putative transposable element of original structure, which was likely involved in its mobilization from the Aeromonas metagenome.

Diversity of capsular polysaccharide gene clusters in Kpc-producing Klebsiella pneumoniae clinical isolates of sequence type 258 involved in the Italian epidemic.

Strains of Klebsiella pneumoniae producing KPC-type beta-lactamases (KPC-Kp) are broadly disseminating worldwide and constitute a major healthcare threat given their extensively drug resistant phenotypes and ability to rapidly disseminate in healthcare settings. In this work we report on the characterization of two different capsular polysaccharide (CPS) gene clusters, named cpsBO-4 and cps207-2, from two KPC-Kp clinical strains from Italy belonging in sequence type (ST) 258, which is one of the most successful ST of KPC-Kp spreading worldwide. While cpsBO-4 was different from known 78 K-types according to the recently proposed typing schemes based on the wzi or wzc gene sequences, cps207-2 was classified as K41 by one of these methods. Bioinformatic analysis revealed that they were represented in the genomic sequences of KPC-Kp from strains of ST258 from different countries, and cpsBO-4 was also detected in a KPC-Kp strain of ST442 from Brazil. Investigation of a collection of 46 ST258 and ST512 (a single locus variant of ST258) clinical strains representative of the recent Italian epidemic of KPC-Kp by means of a multiplex PCR typing approach revealed that cpsBO-4 was the most prevalent type, being detected both in ST258 and ST512 strains with a countrywide distribution, while cps207-2 was only detected in ST258 strains with a more restricted distribution.

Cross-infection of solid organ transplant recipients by a multidrug-resistant Klebsiella pneumoniae isolate producing the OXA-48 carbapenemase, likely derived from a multiorgan donor.

We describe two cases of bacteremic infections caused by a multidrug-resistant Klebsiella pneumoniae isolate producing the OXA-48 carbapenemase that occurred in two solid organ transplant (liver and kidney) recipients, which was apparently transmitted with the allografts. This finding underscores the risk of donor-derived infections by multidrug-resistant Gram-negative pathogens in solid organ transplant recipients and emphasizes the need for rapid screening of organ donors for carriage of similar pathogens.

Rapid resistome fingerprinting and clonal lineage profiling of carbapenem-resistant Klebsiella pneumoniae isolates by targeted next-generation sequencing.

Thirty-two carbapenem-resistant Klebsiella pneumoniae isolates, representative of different resistance mechanisms and clonal lineages, were analyzed with the Pathogenica HAI BioDetection system, based on targeted next-generation sequencing (NGS) technology. With most strains, the system simultaneously yielded comprehensive information on relevant ß-lactam resistance determinants and accurate discrimination of clonal lineages, in a shorter time frame and in a less labor-intensive manner than currently available methods for molecular epidemiology analysis. Results supported the usefulness of targeted NGS-based technologies for similar applications.

Carbapenemase-producing Enterobacteriaceae during 2011-12 in the Bolzano area (Northern Italy): increasing diversity in a low-endemicity setting.

The recent (2011-2012) distribution of carbapenemase determinants in Enterobacteriaceae was studied in the Bolzano area (Northern Italy). Low proportions of carbapenemase producers were found for Escherichia coli (0.2%), Citrobacter freundii (1.1%), Klebsiella pneumoniae (1.3%), Klebsiella oxytoca (1.6%) and Enterobacter spp (1.8%). Although VIM-1 remained the most common carbapenemase, the emergence of K. pneumoniae producing KPC-3 and of E. coli producing OXA-48 was observed. Of concern is the spread of the hyperepidemic strains E. coli ST131 producing VIM-1 and K. pneumoniae ST258 producing KPC-3. Low essential and category agreements between the reference broth microdilution and commercial methods were observed for carbapenems.

Large oligoclonal outbreak due to Klebsiella pneumoniae ST14 and ST26 producing the FOX-7 AmpC ß-lactamase in a neonatal intensive care unit.

A large outbreak caused by expanded-spectrum cephalosporin-resistant Klebsiella pneumoniae (ESCRKP) was observed in a neonatal intensive care unit (NICU) in central Italy. The outbreak involved 127 neonates (99 colonizations and 28 infections, with seven cases of sepsis and two deaths) over a period of more than 2 years (February 2008 to April 2010). Characterization of the 92 nonredundant isolates that were available for further investigation revealed that all of them except one produced the FOX-7 AmpC-type ß-lactamase and belonged to either sequence type 14 (ST14) or ST26. All of the FOX-7-positive isolates were resistant to cefotaxime, ceftazidime, and piperacillin-tazobactam, while 76% were susceptible to cefepime, 98% to ertapenem, 99% to meropenem, and 100% to imipenem. The two carbapenem-nonsusceptible isolates had alterations in the genes encoding outer membrane proteins K35 and K36, which resulted in truncated and likely nonfunctional proteins. The outbreak was eventually controlled by the reinforcement of infection control measures based on a multitiered interventional approach. This is the first report of a large NICU outbreak caused by ESCRKP producing an AmpC-type enzyme. This study demonstrates that AmpC-type enzyme-producing strains can cause large outbreaks with significant morbidity and mortality effects (the mortality rate at 14 days was 28.5% for episodes of sepsis), and it underscores the role of laboratory-based surveillance and infection control measures to contain similar episodes.

Persistent carriage and infection by multidrug-resistant Escherichia coli ST405 producing NDM-1 carbapenemase: report on the first Italian cases.

We report on the first detection of the NDM-1 carbapenemase in Italy, in Escherichia coli isolated in October 2009. Prolonged colonization and relapsing infection by NDM-1-positive E. coli were observed in a patient (index case) with an indirect epidemiological link with areas of endemicity. Transient colonization was apparently observed in another patient linked with the index case.