Lida Politi

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece

OBJECTIVES Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. METHODS Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-β-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. RESULTS Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ∼95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. CONCLUSIONS This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region.

Emergence of OXA-162 Carbapenemase- and DHA-1 AmpC Cephalosporinase-Producing Sequence Type 11 Klebsiella pneumoniae Causing Community-Onset Infection in Greece

ABSTRACT OXA-48-like carbapenemases have only recently emerged in Europe. OXA-162 is a rare OXA-48 variant usually coexpressed with extended-spectrum β-lactamases. Here, we report the identification of the first OXA-162 carbapenemase-producing Klebsiella pneumoniae isolates, which coexpressed an AmpC cephalosporinase (DHA-1), retrieved from a patient in Greece. They belonged to a single sequence type (ST11) and caused the first documented community-onset urinary tract infections attributable to an OXA-48-like-producing Enterobacteriaceae strain.

Epidemiological surveillance of multidrug‐resistant gram‐negative bacteria in a solid organ transplantation department

We assessed the impact of intensified infection control measures (ICM) on colonization and infection caused by carbapenem‐resistant (CR) Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii in a solid organ transplantation (SOT) department.

In Vitro Bactericidal Activity of Trimethoprim-Sulfamethoxazole Alone and in Combination with Colistin against Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates.

ABSTRACT Trimethoprim-sulfamethoxazole alone and combined with colistin was tested in vitro against six carbapenem-resistant Acinetobacter baumannii (CRAB) clinical strains. After 24 h, at achievable serum concentrations, trimethoprim-sulfamethoxazole effectively killed all strains, while colistin killed only one strain. Trimethoprim-sulfamethoxazole plus colistin rapidly killed all strains after 6 h and for up to 24 h. Trimethoprim-sulfamethoxazole, one of the few remaining antimicrobials that still has a degree of activity, particularly combined with colistin, might represent an effective therapy for severe CRAB infections.

First report of an NDM-1 metallo-β-lactamase-producing Acinetobacter baumannii clinical isolate in Greece

Acinetobacter baumannii represents a successful opportunistic pathogen commonly implicated in nosocomial infections. We aim to report the first NDM-1 metallo-β-lactamaseand OXA-23 carbapenemase-producing A. baumannii isolate detected in the Balkans after having been imported to Greece via patient transfer from Libya. In February 2015, a 27-year-old male patient was transferred to Metropolitan General Hospital (Piraeus, Greece) from Libya, where he had been intermittently hospitalised over a 2-month period owing to a contaminated fracture of the left femur. As part of our standing active surveillance protocols for multidrugresistant (MDR) pathogens, a rectal swab was obtained upon admission, yielding a carbapenem-resistant A. baumannii. An open amputation of the middle of the femur was performed within the first day of hospitalisation. Tissue cultures obtained from intraoperative specimens grew a carbapenem-resistant A. baumannii and a carbapenem-susceptible CTX-M-producing Klebsiella pneumoniae isolate. Carbapenem-resistant A. baumanniiwas also isolated from the patient’s tracheal aspirates on Day 9 of hospitalisation, however without any clinical indication of pulmonary infection. Over the following 3-month hospitalisation period, carbapenem-resistant A. baumannii isolates were retrieved from a total of five consecutive wound swabs with or without the additional presence of the K. pneumoniae. Consecutive blood and urine cultures yielded no micro-organisms. Pending and following molecular testing, infection control measures were strengthened by cohorting the patient and reinforcing diligent hand hygiene using an alcohol-based hand rub prior to and after patient handling, using disposable gloves and meticulous disinfecting of all related surfaces and equipment. Species identification was performed using the VITEK®2 system (bioMérieux, Marcy-l’Étoile, France) and was validated by PCR amplification and sequencing of the intrinsic blaOXA-51-like carbapenemase gene. Minimum inhibitory concentrations (MICs) were also determined using the VITEK®2 system and were interpreted according to the 2014 Clinical and Laboratory Standards Institute (CLSI) criteria. Carbapenem MICs were confirmed by Etest (bioMérieux). Tigecycline susceptibility was assessed according to the US Food and Drug Administration (FDA) recommendations for Enterobacteriaceae (susceptible, ≤2mg/L; resistant, ≥8mg/L). The A. baumannii isolates were genotypically identical and exhibited resistance to β-lactams including penicillins, their combinations with inhibitors, expanded-spectrum cephalosporins and carbapenems (imipenem and meropenem MICs ≥16 mg/L); they retained susceptibility to minocycline, tigecycline (MIC ≤ 0.5 mg/L) and colistin (MIC ≤ 2 mg/L). They exhibited resistance to gentamicin and tobramycin but remained susceptible to amikacin. Carbapenemase activity was evaluated by the modified Hodge test (MHT) using a meropenem disk. Screening for class A and B carbapenemases was carried out with combined disk tests using meropenem as substrate without and with phenyl boronic acid (PBA), ethylene diamine tetra-acetic acid (EDTA) or both. The MHT was positive and the combined disk tests were indicative of class B carbapenemase production. Single PCR reactions were used to interrogate the A. baumannii isolate for the presence of OXA-23, OXA-24, OXA-58 and other carbapenemase genes such as NDM, VIM, IMP, KPC, OXA-48-like as well as OXA-1, OXA-2 and extended-spectrum β-lactamases (ESBLs), including SHV, TEM and CTX-M-types. The presence of the ISAba125 element and the bleMBL gene was investigated, and mapping of the blaNDM surrounding environment was performed. The ISAba1 element was also sought in relation to blaOXA carbapenemase genes. Presence of the blaNDM-1, blaOXA-23, blaOXA-51 and blaTEM genes was established. The blaNDM-1 gene was flanked upstream by an ISAba125 element and downstream by the bleMBL gene, whilst the blaOXA-23 gene was flanked upstream by an ISAba1 element. Single-locus blaOXA-51-like sequence-based typing was performed, assigning the isolate to OXA66 of the international clonal complex 2 (CC2) [1]. Meropenem [200 mg every 8 h (q8h) as an extended 4-h intravenous infusion (IVI)], colistin (3 MU q8h IVI), tigecycline [100 mg every 12 h (q12h) IVI] and amikacin (500 mg q12h IVI) were administered. Repeated surgical debridement was performed until the wound gradually healed and wound swab cultures remained negative. Upon discharge, the patient was prescribed amoxicillin/ clavulanic acid and minocycline for an additional 4-week period. Identification of the NDM gene in Acinetobacter spp. represents an additional burden from an infection control standpoint especially due to the existing speculation that it may also act as an NDM gene reservoir for dissemination to other recipient species [2]. Worldwide reports highlight the appearance of such isolates in India, Israel, Egypt, Germany, Spain, Switzerland, the United Arab Emirates and China [2], with a high estimated prevalence in the Indian/Pakistani peninsula, northern Africa [3,4] and the Middle East [5]. In Europe, however, case reports of colonisation and infection remain limited, with several having an established epidemiological link to northern Africa (Algiers, Libya and Egypt) or the Balkans [2]. Certain regions, such as the Indian peninsula and the Balkans, may offer a favourable setting for autochthonous acquisition, nevertheless international travel between countries and continents for medical purposes or not has provided an additional vehicle for the global spread of NDM-producers [2]. This work, however, is an example of how the implementation of diligent surveillance protocols can contain further dissemination even after the introduction of a novel MDR clonal strain due to the current medical globalisation status. Funding: None. Competing interests: None declared. Ethical approval: Not required.

Cluster-distinguishing genotypic and phenotypic diversity of carbapenem-resistant Gram-negative bacteria in solid-organ transplantation patients: a comparative study

Purpose. Solid‐organ transplant recipients may display high rates of colonization and/or infection by multidrug‐resistant bacteria. We analysed and compared the phenotypic and genotypic diversity of carbapenem‐resistant (CR) strains of Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii isolated from patients in the Solid Organ Transplantation department of our hospital. Methodology. Between March 2012 and August 2013, 56 CR strains from various biological fluids underwent antimicrobial susceptibility testing with VITEK 2, molecular analysis by PCR amplification and genotypic analysis with pulsed‐field gel electrophoresis (PFGE). They were clustered according to antimicrobial drug susceptibility and genotypic profiles. Diversity analyses were performed by calculating Simpson’s diversity index and applying computed rarefaction curves. Results/Key findings. Among K. pneumoniae, KP‐producers predominated (57.1%). VIM and OXA‐23 carbapenemases prevailed among P. aeruginosa and A. baumannii (89.4 and 88.9%, respectively). KPC‐producing K. pneumoniae and OXA‐23 A. baumannii were assigned in single PFGE pulsotypes. VIM‐producing P. aeruginosa generated multiple pulsotypes. CR K. pneumoniae strains displayed phenotypic diversity in tigecycline, colistin (CS), amikacin (AMK), gentamicin (GEN) and co‐trimoxazole (SXT) (16 clusters); P. aeruginosa displayed phenotypic diversity in cefepime (FEP), ceftazidime, aztreonam, piperacillin, piperacillin‐tazobactam, AMK, GEN and CS (9 clusters); and A. baumannii displayed phenotypic diversity in AMK, GEN, SXT, FEP, tobramycin and rifampicin (8 clusters). The Simpson diversity indices for the interpretative phenotype and PFGE analysis were 0.89 and 0.6, respectively, for K. pneumoniae strains (P<0.001); 0.77 and 0.6 for P. aeruginosa (P=0.22); and 0.86 and 0.19 for A. baumannii (P=0.004). Conclusion. The presence of different antimicrobial susceptibility profiles does not preclude the possibility that two CR K. pneumoniae or A. baumannii isolates are clonally related.

Colistin Resistance in KPC-2- and SHV-5-Producing Klebsiella pneumoniae Clinical Isolates in Bulgaria

Background/Aims: Colistin resistance is increasingly recognized among carbapenemase-producing Klebsiella pneumoniae isolates in several European regions. The current study documents the appearance of colistin resistance among KPC-2 and SHV-5-produning K. pneumoniae strains in Bulgaria. Methods: Four colistin-resistant K. pneumoniae isolates were recovered from 2 patients hospitalized in the anesthesiology and resuscitation clinic of a tertiary care university hospital in Sofia, Bulgaria. Microbial identification and antimicrobial susceptibility testing was performed by Vitek 2 (Biomerieux, France). β-Lactamase genes were amplified using a panel of primers for detection of all MBL-types, KPCs, plasmid-mediated AmpCs in single PCR reactions, OXA-type carbapenemases, extended-spectrum β-lactamases (ESBLs) and TEM enzymes. The colistin-resistant mcr-1 gene was also investigated using previously described primers and conditions. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to investigate clonality. Results: The 4 K. pneumoniae isolates exhibited colistin MICs >16 mg/L and showed multidrug-resistant phenotypes, remaining intermediately susceptible only to gentamicin. They were clustered into a single PFGE clonal type and MLST assigned them to sequence type 258. All isolates possessed KPC-2 carbapenemase and SHV-5 ESBL. They were negative for the plasmid-mediated colistin-resistant mcr-1 gene, possibly implying an intrinsic mechanism of resistance. Conclusions: Although colistin use in Bulgaria only started moderately during 2014, the findings of the current study notify the appearance of colistin resistance among carbapenemase-producing Klebsiella species in another European region.

Absence of human rhinovirus and respiratory syncytial virus from bronchoalveolar lavage and bronchial biopsies of selected patients with stable chronic obstructive pulmonary disease

Previous studies have reported very different rates of human rhinovirus (HRV) and respiratory syncytial virus (RSV) genome detection in nasal and sputum samples, but not in bronchoalveolar lavage (BAL) and bronchial biopsy samples. Our study aimed to investigate the presence of HRV and RSV in the lungs of 31 consecutive patients with stable COPD (11 GOLD stage I, 11 II, and 9 III) and 22 control subjects (12 current or past smokers, and 10 non-smokers), who underwent diagnostic (e.g., lung cancer) and/or therapeutic (e.g., hemoptysis) fibreoptic bronchoscopy in a university hospital in Athens, Greece. Viral RNA of HRV and RSV were not detected in any of the samples of COPD patients or control subjects after being processed with real-time PCR.

Hospital outbreak due to a Klebsiella pneumoniae ST147 clonal strain co-producing KPC-2 and VIM-1 carbapenemases in a tertiary teaching hospital in Northern Greece

Here we present the characteristics of a large outbreak caused by a clonal Klebsiella pneumoniae strain producing both KPC-2 and VIM-1 carbapenemases in a tertiary teaching hospital. Between January 2013 and January 2015, 45 carbapenem-resistant K. pneumoniae isolates that gave a positive modified Hodge test and were phenotypically suspected of metallo-β-lactamase (MBL) and K. pneumoniae carbapenemase (KPC) co-production were recovered from 25 patients hospitalised in AHEPA University Hospital (Thessaloniki, Greece). All of the patients were hospitalised in the three intensive care units of the hospital and 17 (68%) of them developed bloodstream infections; the overall mortality of the patients involved in the outbreak was 48% (12/25). Molecular testing verified that all 45 K. pneumoniae isolates co-harboured blaKPC-2 and blaVIM-1 genes and were associated with OmpK35 deficiency and OmpK36 porin loss. The blaTEM-1 gene was also present in 18 isolates. Pulsed-field gel electrophoresis (PFGE) clustered all of the isolates into a single clonal type, and multilocus sequence typing (MLST) assigned them to the emerging high-risk ST147 clonal lineage. Following recognition of the outbreak, infection control measures were implemented in the affected areas. The outbreak continued for ca. 2 years and since then only sporadic cases of K. pneumoniae harbouring both carbapenemases have been detected.

Characterization of extensively- or pandrug-resistant ST147 and ST101 OXA-48-producing Klebsiella pneumoniae isolates causing bloodstream infections in ICU patients

ABSTRACT 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.