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We characterized Staphylococcus aureus strains isolated from cystic fibrosis (CF) patients during screening for multidrug-resistant strains to determine mechanisms of antibiotic resistance and cond...

Bloodstream infections (BSIs) after chemotherapy or hematopoietic stem cell transplantation (HSCT) are a leading cause of morbidity and mortality. Data on 154 BSIs that occurred in 111 onco-hematological patients (57 hematological malignancies, 28 solid tumors, and 26 non-malignant hematological diseases) were retrospectively collected and analyzed. Monomicrobial Gram-positive (GP), Gram-negative (GN), and fungal BSIs accounted for 50% (77/154), 38.3% (59/144), and 3.2% (5/154) of all episodes. Polymicrobial infections were 7.8% (12/154), while mixed bacterial–fungal infections were 0.6% (1/154). The most frequent GN isolates were Escherichia coli (46.9%), followed by Pseudomonas aeruginosa (21.9%), Klebsiella species (18.8%), and Enterobacter species (6.3%). Overall, 18.8% (12/64) of GN organisms were multidrug-resistant (seven Escherichia coli, three Klebsiella pneumoniae, and two Enterobacter cloacae), whereas GP resistance to glycopeptides was observed in 1% (1/97). Initial empirical antibiotic therapy was deemed inappropriate in 12.3% of BSIs (19/154). The 30-day mortality was 7.1% (11/154), while the bacteremia-attributable mortality was 3.9% (6/154). In multivariate analysis, septic shock was significantly associated with 30-day mortality (p = 0.0001). Attentive analysis of epidemiology and continuous microbiological surveillance are essential for the appropriate treatment of bacterial infections in pediatric onco-hematological patients.

ObjectiveTo investigate the presence of bacteria and fungi in bronchial aspirate (BA) samples from 43 mechanically ventilated patients with severe COVID-19 disease.MethodsDetection of SARS-CoV-2 was performed using Allplex 2019-nCoV assay kits. Isolation and characterisation of bacteria and fungi were carried out in BA specimens treated with 1X dithiothreitol 1% for 30 min at room temperature, using standard culture procedures.ResultsBacterial and/or fungal superinfection was detected in 25 out of 43 mechanically ventilated patients, generally after 7 days of hospitalisation in an intensive care unit (ICU). Microbial colonisation (colony forming units (CFU) Pseudomonas aeruginosa was identified in 17 patients. Interestingly, 11 out of these 17 isolates also showed carbapenem resistance. The molecular analysis demonstrated that resistance to carbapenems was primarily related to OprD mutation or deletion. Klebsiella pneumoniae was the second most isolated pathogen found in 13 samples, of which 8 were carbapenemase-producer strains.ConclusionThese data demonstrate the detection of bacterial superinfection and antimicrobial resistance in severe SARS-CoV-2-infected patients and suggest that bacteria may play an important role in COVID-19 evolution. A prospective study is needed to verify the incidence of bacterial and fungal infections and their influence on the health outcomes of COVID-19 patients.

This study focused on Klebsiella pneumoniae isolates that were resistant or had low susceptibility to a combination of ceftazidime/avibactam. We aimed to investigate the mechanisms underlying this resistance. A total of 24 multi-drug resistant isolates of K. pneumoniae were included in the study. The phenotypic determination of carbapenemase presence was based on the CARBA NP test. NG-Test CARBA 5 was also performed, and it showed KPC production in 22 out 24 strains. The molecular characterisation of blaKPC carbapenemase gene, ESBL genes (blaCTX-M, blaTEM, and blaSHV) and porin genes ompK35/36 was performed using the PCR. Finally, ILLUMINA sequencing was performed to determine the presence of genetic mutations.Various types of mutations in the KPC sequence, leading to ceftazidime/avibactam resistance, were detected in the analysed resistant strains. We observed that KPC-31 harboured the D179Y mutation, the deletion of the amino acids 167–168, and the mutation of T243M associated with ceftazidime/avibactam resistance. The isolates that did not present carbapenemase alterations were found to have other mechanisms such as mutations in the porins. The mutations both on the KPC-3 enzyme and in the porins confirmed, that diverse mechanisms confer resistance to ceftazidime/avibactam in K. pneumoniae.

Background: This study aimed to evaluate the effectiveness of piperacillin–tazobactam as antibiotic prophylaxis in patients affected by a peri-ampullary tumor submitted to pancreatic surgery. Metho...

Since the beginning of the SARS-CoV-2 pandemic, COVID-19 appeared as a unique disease with unconventional tissue and systemic immune features. Here we show a COVID-19 immune signature associated with severity by integrating single-cell RNA-seq analysis from blood samples and broncho-alveolar lavage fluids with clinical, immunological and functional ex vivo data. This signature is characterized by lung accumulation of naïve lymphoid cells associated with a systemic expansion and activation of myeloid cells. Myeloid-driven immune suppression is a hallmark of COVID-19 evolution, highlighting arginase-1 expression with immune regulatory features of monocytes. Monocyte-dependent and neutrophil-dependent immune suppression loss is associated with fatal clinical outcome in severe patients. Additionally, our analysis shows a lung CXCR6+ effector memory T cell subset is associated with better prognosis in patients with severe COVID-19. In summary, COVID-19-induced myeloid dysregulation and lymphoid impairment establish a condition of ‘immune silence’ in patients with critical COVID-19., Integrated studies of matched tissue sites and cell types in COVID-19 patients are important to define the immune mechanisms of pathology. Here, the authors describe an immune signature in fatal COVID-19 patients harmonizing single-cell RNA sequencing of blood and matched BAL cells with deep clinical, immunological and functional data.

Rapid detection of Methicillin Resistant Staphylococcus aureus (MRSA) is an important concern for both treatment and implementation of infection control policies. The present study provides an ‘in house’ real-time PCR assay to detect directly nuc, pvl, and mecA genes. The assay is able to perform identification of MRSA, Methicillin-Sensitive S. aureus, Methicillin-Resistant Coagulase Negative Staphylococci and the Panton-Valentine leukocidin virulence gene from rectal and pharyngeal swab samples in a screening context. We found an analytical sensitivity of this current Triplex PCR assay of 514 CFU/mL. Analytical specificity was tested with different Gram-positive and Gram-negative species and yielded no false-positive PCR signal. The sensitivity and specificity of the Triplex Real Time PCR were both 100% for these targets when compared with the culture and conventional methods. This assay is readily adaptable for routine use in a microbiology laboratory, as it will enable the implementation of timely and properly guided therapy and infection control strategies.

Clinical microbiology laboratories in hospital settings need to be able to identify patients who carry carbapenemase-producing bacterial strains quickly in order to contain their spread and initiate proper pharmacological therapy. The aim of this study was to confirm the correlation between KPC production and a characteristic mass spectrometry (MS) peak (11 109 Da±8) to validate the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS as a rapid screening tool. With this aim, 176 selected clinical samples that were KPC-producing and 260 control samples that were carbapenem-susceptible or carbapenem-resistant through other resistance mechanisms, or were producing hydrolytic enzymes other than KPC, were analysed. The presence of the 11 109 Da peak in the spectra of 99.4 % (175/176) of the KPC-producing strains compared to the controls, which all lacked the peak, confirmed a strong correlation between KPC production and the presence of the 11 109 Da peak in the MALDI-TOF MS spectrum. The high sensitivity (98.7 %) and specificity (100 %) of the peak searching in the MALDI-TOF MS spectra mean that 11 109 Da peak searching is a suitable screening tool in KPC-endemic regions.

Recently, Escherichia coli producing extended-spectrum β-lactamases (ESBLs) have become a serious public-health problem, and food-producing animals (FPAs) have been suggested as a potential reservoir/source. This study aimed to compare ESBL-producing E. coli isolates from different sources. ESBL-producing E. coli isolates were collected from humans (n = 480) and FPAs (n = 445) in Italy (2016–2017). Isolates were screened for the presence of ESBL and carbapenemase genes and were classified according to phylogenetic group and MLST genotyping. The genes mcr-1 to -5 were searched for in colistin-resistant isolates. CTX-M was the most frequent ESBL type both in human and animal isolates. CTX-M-15 prevailed in humans (75.0%) and cattle (51.1%) but not in poultry (36.6%). CTX-M-1 was common (58.3%) in pigs. SHV-type and CMY-2-like were found in FPAs, especially in poultry (17.0% and 29.9%, respectively). Additionally, 29 isolates were mcr-1 carriers (3 from humans and 26 from FPAs). No carbapenemase genes were detected. Human isolates mostly belonged to phylogroup B2 (76.5%). Animal isolates were distributed among groups A (35.7%), B1 (26.1%) and C (12.4%). Few animal isolates (almost all from poultry) were classified into group B2 (4.3%). Most human isolates (83.4%) belonged to the pandemic ST131 clone and frequently carried CTX-M-15 (75.9%). ST131 was rarely detected in FPAs (three isolates from poultry). Nineteen STs were shared in both sources, with ST10, ST410 and ST69 being more frequently detected. Potential exchange of ESBL genes from animals to humans is feasible, underlying the need for strict monitoring based on a ‘One Health’ approach.

NorA is a Staphylococcus aureus multidrug transporter that exports structurally distinct compounds including fluoroquinolones. In this study norA-like genes of Staphylococcus epidermidis (norASEP) and Staphylococcus haemolyticus (norASHAE) were identified and sequenced. The nucleotide identity of norASEP and norASHAE with norA was 75.3 and 74.1%, respectively, and the amino acid identity 87.7 and 86%, respectively. Inactivation of norASEP increased the ciprofloxacin susceptibility of E. coli DH5α carrying the pB SK 198 norASEP EZ cat norASEP plasmid.

Objectives Carbapenemase-producing strains of Klebsiella pneumoniae (KPC) are a great health concern, and therapy with ceftazidime–avibactam represents a choice for the treatment of infections involving these strains. We report a strain resistant to ceftazidime–avibactam due to a deletion of six nucleotides in the blaKPC gene sequence. Methods Two strains, namely AMP920 and AMP2009, were isolated from the same patient a month apart. Antimicrobial susceptibility testing was performed both by broth microdilution and by Etest. Immunoenzymatic assay to detect carbapenemase was performed for both strains. The blaKPC gene of both strains was amplified by PCR and sequenced. Enzyme activity towards carbapenems was tested by the CarbaNP test and hydrolysis spectrophotometer assay. Results The two isolates differed in antimicrobial susceptibility. AMP920 showed meropenem and imipenem resistance (MIC 32 and 32 mg/mL). A month later the carbapenem MIC decreased to 8 and 1 mg/mL respectively, while the ceftazidime–avibactam MIC increased from 1 to 16 mg/mL. Both isolates showed a positive immunoenzymatic test for the KPC enzyme, but only AMP920 showed a positive CarbaNP test hydrolysing imipenem. The BlaKPC gene was amplified in both strains. After sequencing, the two amplicons showed a KPC3 variant. The gene of the second isolate showed a deletion of six nucleotides at 498–503, resulting in a mutant variant with the deletion of glutamic acid and leucine residues at positions 167 and 168. Conclusions We detected a new deletion in the blaKPC gene of a clinical strain of K. pneumoniae which resulted in resistance to ceftazidime–avibactam. The amino acids deleted are in the Ω loop (amino acids 165–179) of the KPC enzyme, enhancing ceftazidime affinity and preventing avibactam binding.

Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Vitek MS system is a useful technique to identify bacteria strains isolated in clinical samples. In this paper, we applied this method to KPC-producing Enterobacteriaceae detection through the determination of a specific 11,109 (±8) Da peak. We assayed the presence, specificity and reliability of this peak on routine workflow through the analysis of 183 Enterobacteriaceae strains isolated from clinical samples and characterized by classical approaches. The peak was detected in 95.5% (129/135) of carbapenemase-producing strains spectra compared with the 48 extended spectrum beta-lactamase producing controls strains, which all lacked this peak. Hence, this 11,109 Da peak determination showed a Positive Predictive Value (PPV) of 100% and a Negative Predictive Value (NPV) of 94.4%. The characterization of this specific peak in a MALDI-TOF Vitek MS system might be considered a valuable tool to reveal KPC-producing Enterobacteriaceae especially in KPC endemic region.

Background Despite improvements in the perioperative care, the morbidity rate after pancreaticoduodenectomy (PD) is still higher than 50%. The aim of this study was twofold: first, to assess the correlation between preoperative rectal swab (RS) and intraoperative bile cultures; to examine the impact of RS isolates on postoperative course after PD. Methods An observational study was conducted analyzing all consecutive PD performed from January 2015 to July 2016. Based on the positivity/negativity of preoperative RS for multi-drug resistant bacteria, two groups of patients were identified (RS+ vs. RS−) and then compared. Results Three hundred thirty-eight patients were considered for the analysis. RS culture showed a perfect correlation (species and phenotypic antibiotic susceptibility pattern) with bile culture in 157 patients (86.7%). Fifty patients (14.8%) had a RS+. Preoperative biliary drain (PBD) was the single independent preoperative risk factor associated to RS+ (p = 0.021, OR = 2.6, 95% CI = 1.5–11.7). Infective complications (IC) and mortality were independently correlated to RS+ (p = 0.013, OR = 2.9, 95% CI = 1.3–6.7; p = 0.009 OR = 3.4, 95% CI = 1.8–14.9, respectively). Conclusions Preoperative surveillance RS-culture's positivity correlates to biliary colonization that occurs after PBD. IC and mortality after PD are associated with RS+. Preoperative RS can direct antibiotic prophylaxis to reduce morbidity and mortality after PD.

Since the first carbapenem-resistant Klebsiella pneumoniae strain was isolated in 2008, Enterobacteriaceae with reduced susceptibility to one or more carbapenems have emerged sporadically in different geographical regions in Croatia. These observations gave rise to a multicenter study on carbapenem resistance in Enterobacteriaceae from Croatia. Fifty-seven carbapenem-non-susceptible strains of Enterobacteriaceae were collected during 2011–2012 from four large hospital centres in Croatia. Overall, 36 strains produced VIM-1 β-lactamase, three produced NDM-1, and one produced KPC-2. A high degree of clonal relatedness was observed in Enterobacter cloacae and Citrobacter freundii strains, in contrast to K. pneumoniae strains. Bla VIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aac A4 ). The study found strong association between bla VIM and qnr B6 and between bla NDM and qnr A6 genes.

Urinary tract infections (UTIs) are among the most frequent infectious diseases affecting humans, and represent an important public health problem with a substantial economic burden. Due to the high empiric use of antibiotics for the treatment of UTI, antibacterial resistance of Enterobacteriaceae, specifically the main uropathogens Escherichia coli and Klebsiella pneumoniae, has significantly increased worldwide. In this article the worldwide epidemiology of resistant Gram-negative bacteria causing UTIs, with a special focus on extended spectrum beta lactamase (ESBL) positive pathogens, as well as new threats such as multi-drug-resistant (MDR) clones (e.g. E. coli 131 (ST131) and K. pneumoniae ST258), are reviewed. The increased prevalence of MDR Enterobacteriaceae, limiting available treatment options for infections caused by these organisms, and the lack of new antibiotics provide good rationale for using older antibiotics, such as fosfomycin, that have been shown to retain some activity against MDR bacteria.

We investigated the presence of β-lactamase genes (bla) in 26 strains of Enterobacteriaceae already found positive for the qnrS1 gene, a plasmid-mediated quinolone resistance determinant. Three strains of K. pneumoniae, isolated in the period 2008-2009 at the University Hospital in Verona, were positive for LAP-2, a narrow-spectrum β-lactamase. These strains, namely VRB586, VRE185 and VRE196, were cultured from urine, bile and peritoneal drainage, respectively, of different patients from different units. The bla(LAP-2) and qnrS1 resistance determinant genes were separated by ISEcl2 and were located on a 97 kb conjugable and untypable plasmid, which could be transferred to a recipient strain, E. coli J53. The fluoroquinolone and ceftazidime MICs increased 1-2-fold in the transconjugant cells. The three K. pneumoniae strains were found to be clonal by PFGE and were identified as belonging to ST147, an internationally successful clone, by MLST. The plasmid sequence, including ISEcl2 and qnrS1 genes, of K. pneumoniae ST147 was found to be highly similar to previously detected qnrS1-harbouring plasmids, suggesting the plasmid has a stable genetic structure and that these resistance determinants have a common source. To the best of our knowledge, this is the first report of the internationally successful K. pneumoniae ST147 strain carrying bla(LAP-2) and qnrS1 genes and is the first case of LAP β-lactamase in Italy.

The rapid emergence of KPC-producing Klebsiella pneumoniae has become a serious problem in health-care settings, increasing in frequency worldwide. These infections are worrisome, since the antimicrobial treatment options for infections due to multidrug-resistant strains are very limited, and outbreaks must be rapidly detected and controlled. A semi-automated, repetitive-sequence-based PCR (rep-PCR) instrument (DiversiLab system) was evaluated in comparison with the pulse-field gel electrophoresis (PFGE) and multilocus sequence typing to investigate the outbreak of KPC-producing K. pneumoniae in a surgery unit at the University Hospital of Verona, Italy, as a rapid method for outbreak investigations. A selection of seven epidemiologically related K. pneumoniae showing resistance to carbapenem and three epidemiologically unrelated K. pneumoniae isolates were collected from patient with hospital-acquired infection. Among the epidemiologically related isolates, PFGE and Rep-PCR identified a unique pattern with more than 90% of homology. The concordance between DiversiLab and PFGE results confirmed the usefulness of rapid molecular techniques to investigate outbreaks due to multidrug-resistant bacteria. Moreover, this result could meet the international need for a harmonised typing tool, allowing the implementation of strict control measures to prevent dissemination of these organisms in health-care settings.

We report an outbreak of linezolid-resistant Staphylococcus haemolyticus strains (MIC 32 mg/L) in patients admitted to the Verona University Hospital Intensive Care Unit. The strains proved to be clonally related at pulsed field gel electrophoresis. All the strains showed the G2576T mutation responsible for linezolid-resistance and retained their resistance even after several passages on antibiotic-free medium. After a decade of linezolid use, multifocal emergence of linezolid resistance in coagulase-negative staphylococci has become an important matter of concern and mandates stricter control over the use of this antibiotic in order to preserve its clinical utility.

Four NDM-1-producing Enterobacteriaceae strains (three Klebsiella pneumoniae and one Citrobacter koseri) were isolated between 2009 and 2011 through a nationwide surveillance for carbapenem- resistant Enterobacteriaceae in Croatia to study the molecular genetic background of blaNDM and the responsible plasmid types. Phenotypically, the clinical strains proved to be multidrug resistant. All strains remained susceptible to tigecycline and colistin. The clinical strains harbored variable antibiotic resistance determinants, notably, blaNDM-1, blaTEM-1, blaSHV-1, blaSHV-12, blaOXA-1, blaOXA-9, blaCTX-M-15, blaCMY-4, qnrB1, and aac(6')Ib-cr in different combinations. Two K. pneumoniae belonged to sequence type ST15 and one strain to ST16. As for the plasmid types, C. koseri and one of the ST15 K. pneumoniae carried IncR, and the second ST15 K. pneumoniae carried IncR and colE. The K. pneumoniae ST16 strain hosted A/C and colE plasmids. The blaNDM-1 gene was detected on conjugative high-molecular-weight plasmids, namely, A/C and IncR types. It is noteworthy that this is the first description of K. pneumoniae ST16 expressing NDM-1 in Europe. Remarkably, our study underscores the importance of the IncR plasmid as a reservoir of multidrug resistance. To the best of our knowledge, the IncR plasmid carrying blaNDM-1 in C. koseri is reported for the first time.

A carbapenem-resistant Klebsiella pneumoniae was isolated from a blood-culture of an inpatient from Libya, hospitalized in the intensive-care unit of Negrar Hospital, Italy. The clinical isolate carried the following β-lactamase genes, bla TEM-1 , bla SHV-11 , bla OXA-1 , bla CTX-M-15 and bla OXA-48 , respectively. The bla OXA-48 gene was inserted in the Tn 1999.2 transposon type, carried on a conjugative, 60-kilobase plasmid, that presented an L/M backbone, hosted by a multidrug-resistant ST 101 K. pneumoniae strain. Our report highlights the international transfer of bla OXA-48 gene and the importance of screening measures of multidrug-resistant Enterobacteriaceae .

To the Editor: The novel metallo-β-lactamase named New Delhi metallo-β-lactamase (NDM-1) was identified from Klebsiella pneumoniae and Escherichia coli isolates in Sweden from a patient previously hospitalized in India (1). NDM-1 is spreading rapidly worldwide to nonclonally related isolates, many of which are directly or indirectly tracked to the Indian subcontinent (2). A carbapenem-resistant K. pneumoniae strain, KLZA, was isolated in May 2009 from the culture of a blood sample from of a 40-year-old man on the day after his admission to a surgical intensive care unit of the Clinical Hospital Center in Zagreb, Croatia. The patient had been transferred after 5 days of hospitalization in Bosnia and Herzegovina following a car accident. The clinical history mentioned antimicrobial drug treatment that did not include carbapenems (gentamicin, metronidazole, and ceftriaxone) and no link to the Indian subcontinent. Antimicrobial drug susceptibility testing was performed by Vitek2 (bioMerieux, Marcy-l’Etoile, France) and broth microdilution and interpreted according to the latest documents from the European Committee on Antimicrobial Susceptibility Testing (www.eucast.org/clinical_breakpoints/, version 1.1). The strain proved resistant to imipenem and meropenem, to all broad-spectrum cephalosporins, and to aminoglycosides and susceptible to ciprofloxacin and tigecycline (Table). We checked for blaVIM, blaIMP, blaSPM, blaGIM, blaSIM, and blaNDM resistance genes by using PCR. A PCR product was obtained only with the NDM primers, after being purified (QIAquick PCR Purification Kit, QIAGEN, Hilden, Germany), its sequence showed 100% identity with blaNDM-1. Table MIC of the KLZA strain of Klebsiella pneumoniae and its transconjugant and recipient Strain genotyping was performed by multilocus sequence typing to determine the sequence type (ST) of the isolate and to establish a comparison with previously reported NDM-1–producing isolates. Allelic numbers were obtained on the basis of sequences of 7 housekeeping genes at www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html. Multilocus sequence typing identified K. pneumoniae KLZA as an ST25 strain, which significantly differs from the ST14 type found in the index NDM-1–producing strain and from other isolates originating from India (1) and then in other countries. ST25 K. pneumoniae was also found in K. pneumoniae isolates in Geneva (3). Other K. pneumoniae STs harboring NDM-1 were ST15, ST16, and ST147 (4–7). Resistance was transferred by conjugation to E. coli J53, with selection based on growth on agar in the presence of ceftazidime (10 mg/L) and azide (100 mg/L). The conjugant T1 showed resistance to β-lactams, including all carbapenems, as well as decreased susceptibility to ciprofloxacin. The KLZA strain and its transconjugant harbored other determinant of resistance, namely blaCTX-M-15, blaCMY-16, and qnrA6. Plasmid incompatibility groups, determined by a PCR-based replicon typing method, belonged to the incA/C replicon type. This report of an NDM-1–producing K. pneumoniae in Croatia adds to those of other cases in patients from patients hospitalized in the Balkan area. The patient in this report had no apparent link to the Indian subcontinent. In a survey conducted by the European Centre for Disease Prevention and Control to gather information about the spread of NDM-1–producing Enterobacteriaceae in Europe and reporting cases from 13 countries during 2008–2010, five of the 55 persons with known travel histories had traveled to the Balkan region during the month before diagnosis of their infection: 2 to Kosovo and 1 each to Serbia, Montenegro, and Bosnia and Herzegovina. All had received hospital care in Balkan countries because of an illness or accident that occurred during the journey (7). Two of the latter cases (4,8) and a case from Germany (9) were subsequently published. No patient had any apparent link to the Indian subcontinent. Although the way NDM-1 isolates might have been imported to western Europe not only from the Indian subcontinent but also from Balkan countries (10) has been highlighted, awareness of western Europe as a possible area of endemicity remains limited. The aforementioned report from Germany, although recognizing that the patient had been repatriated after hospitalization in Serbia, declared “no evidence about contact with people from regions where NDM-enterobacteria are endemic” (9). This limited awareness shows the threat of neglecting to screen patients who are transferred from countries thought not to be at risk for NDM-1. Furthermore, it means that specimen are not sent to the local reference laboratories and recognized as positive for NDM-1, thus permitting wide dissemination of NDM-1–producing enterobacteria in the community (4). The accumulating evidence of NDM-1 from the Balkan area could suggest a possible multifocal spread of this enzyme, with the Balkans as a possible second area of endemicity, in addition to the Indian subcontinent, and prompts for widespread epidemiologic surveillance.

We investigated the presence of qnrC and qnrD among 756 non-replicate Enterobacteriaceae isolated in Italy, selected for being non-susceptible to fluoroquinolones and/or resistant to third-generation cephalosporins. Four Proteus mirabilis and one Morganella morganii (0.66% of the total) presented a qnrD gene, located in a 2687-base-pair plasmid that was entirely sequenced. The plasmid is un-typable, and contains no known coding region other than qnrD. That the qnrD gene was found in four unrelated P. mirabilis and in one M. morganii isolate might suggest a frequent association of this gene with the tribe Proteeae.

A Pseudomonas aeruginosa highly resistant to carbapenems was isolated in a neonatal intensive care unit in Palermo, Italy. The strain was found to carry a novel VIM‐type enzyme, classified as VIM‐14. The novel enzyme differs from VIM‐4 in a G31S mutation. VIM‐14 was harboured in a class 1 integron with a new organization. The integron carried the genes aac7, blaVIM‐14, blaOXA‐20 and aac4 in that order.

The roles of the AmpC chromosomal β-lactamase and the AcrAB efflux system in levels of intrinsic resistance and susceptibility of Escherichia coli to β-lactams were studied with a set of isogenic strains. MICs of ureidopenicillins, carbenicillin, oxacillin, and cloxacillin were drastically reduced by the inactivation of AcrAB, whereas those of the earlier cephalosporins were affected mostly by the loss of AmpC β-lactamase.

Production of a metallo-β-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-β-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B β-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the β-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to bla IMP , bla VIM was also found to be carried on a gene cassette inserted into a class 1 integron. The bla VIM -containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-β-lactamase-encoding determinant was not transferable to E. coli by conjugation. Expression of the integron-borne bla VIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

A survey of antimicrobial resistance in Streptococcus pyogenes, performed within the framework of a national surveillance program, has revealed a dramatic increase in resistance of S. pyogenes to erythromycin in most areas of Italy. In virtually all the centers that provided data for 3 consecutive years, the incidence of erythromycin-resistant strains increased twofold to 20-fold from 1993 to 1995 and was greater than 30% in five of the 14 centers participating in the study. The clonality of erythromycin-resistant isolates was studied in 15 strains isolated from different patients at the Institute of Microbiology of Verona University (Verona). The features of the Verona isolates and the substantially different rates of erythromycin and clindamycin resistance observed in most centers suggest that the spread of different resistance genes in multiple clones might be occurring throughout the country.

Almost all European countries are affected by the expansion of carbapenem-resistant Enterobacteriaceae occurring during recent years. In the two hospitals of Verona, Italy, the incidence of carbapenem-nonsusceptible Klebsiella pneumoniae (CNSKP) began to increase by the first months of 2011, reached a peak in the summer of the same year, and currently is around 30%. Contrary to what was reported by other hospitals and although significant percentages of CNSKP were detected in respiratory samples, blood and pus, urine from hospitalized patients, mainly geriatrics, are the clinical samples with the highest incidence of these strains. Elder patients are frequently transferred from the hospital to their own homes or long-term care facilities and vice-versa. Moreover, urinary tract infections are not considered as a severe pathology and frequently is asymptomatic in elderly. For these reasons, the presence of carbapenem non-susceptible bacteria in the urinary tract of geriatric patients might be an underestimated cause of multiresistant strain spreading to the non-hospitalized population and the community.

We investigated the prevalence of plasmid-mediated quinolone resistance genes in 756 clinical isolates of Enterobacteriaceae originating from Microbiology Diagnostic Laboratories of North-East Italy. Five point zero two percent of isolates carried a qnr determinant while the aac(6')-Ib-cr determinant was detected in 9·25% of isolates. We also investigated the association between the plasmid-mediated quinolone resistance and the beta-lactamase genes, and characterized the plasmids carrying these determinants of resistance.

The aim of this study was to determine the accuracy of commercial systems (VITEK® 2, Etest and Sensititre®) in determining the minimum inhibitory concentrations of vancomycin, teicoplanin and linezolid of Staphylococcus aureus strains and to evaluate the reproducibility of each system in a clinical microbiology laboratory. In total, 115 strains of S. aureus isolated from blood cultures were tested with all three commercial methods as well as the broth microdilution method, which is designated as the standard for glycopeptides and linezolid. Fourteen different S. aureus strains were included in a reproducibility test for all methods and antibiotics. For these strains, antimicrobial susceptibility testing was repeated 10 times on different days with all four methods, each time using the same inoculum. All three commercial methods exhibited similar performance in categorisation of nearly all of the meticillin-susceptible S. aureus (MSSA) isolates. Discrepancies were registered for meticillin-resistant S. aureus (MRSA); 2.5% of the strains in the intermediate or resistant category with the VITEK 2 system were not recognised as resistant by Etest and Sensititre. Moreover, none of the three commercial methods provided accurate results compared with homemade broth microdilution. Reproducibility of vancomycin and teicoplanin was 100% with VITEK 2 and Sensititre and 98.75% with Etest. Microdilution showed a reproducibility of 95.6% with vancomycin and 83.1% with teicoplanin. In contrast to previous reports, the best agreement with microdilution was exhibited by VITEK 2 both for MSSA and MRSA. For the antibiotics tested, the best reproducibility was obtained with the VITEK 2 and Sensititre systems.

In February 2011, a 78-year-old male patient was admitted to Clinical Hospital Center Zagreb with subdural haematoma. Klebsiella pneumoniae with reduced susceptibility to carbapenems was isolated. PCR revealed the presence of bla(KPC), bla(TEM), and bla(SHV) genes. Sequencing of bla(KPC) gene identified K. pneumoniae carbapenemase (KPC)-2 beta-lactamase. The strain belonged to ST37 clone by multilocus sequence typing. Infection control efforts limited the spread of KPC-producing clone of K. pneumoniae in our hospital so far. To our knowledge, this is the first report of a KPC-producing K. pneumoniae in Croatia.

Linezolid resistance among Gram-positive pathogens is being reported with increasing frequency. We examined 14 linezolid-resistant coagulase-negative staphylococci (CoNS) isolated from blood cultures obtained from patients admitted to the Intensive Care Unit of Vicenza General Hospital, Italy. The species identification yielded 10 Staphylococcus epidermidis, 3 Staphylococcus hominis, and 1 Staphylococcus capitis. Minimal inhibitory concentrations of linezolid ranged between 16 and 32 mg/l. By sequencing domain V of the 23S rRNA gene, 4 isolates were found to harbour a G2576T mutation and 10 isolates a G2447T mutation. None of the strains under study presented either the cfr gene or cardinal mutations in the L3, L4, or L22 riboproteins. In this clinical collection of linezolid-resistant CoNS the G2447T mutation was dominantly associated with S. epidermidis, while the G2576T mutation was found in other CoNS species. Two different CoNS species endowed with either mutation were isolated from 2 patients.