Search

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.

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.

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.

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 .

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.

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.

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.

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.

The worldwide spread of acquired metallo-β-lactamases (MBLs) in gram-negative bacilli has become a great concern. MBLs possess a broad hydrolysis profile that includes carbapenems and almost all extended-spectrum β-lactams. The aim of this investigation was to characterize MBLs in P. aeruginosa isolates from Zagreb, Croatia. 100 P. aeruginosa isolates with reduced susceptibility to either imipenem or meropenem were tested for the production of MBLs by E test MBL. The susceptibility to a wide range of antibiotics was determined by broth microdilution method. The presence of blaMBL genes was detected by PCR. Hydrolysis of 0.1 mM imipenem by crude enzyme preparations of β-lactamases was monitored by UV spectrophotometer.. Outer membrane proteins were prepared and analysed by SDS-PAGE. Six out of 100 isolates were positive for MBLs by E test. All strains were resistant to gentamicin, ceftazidime and cefotaxime and all except of one to imipenem. Six strains positive for MBLs in E test were identified as VIM MBLs producers by PCR. Sequencing of blaVIM genes revealed the production of VIM-2 β -lactamase in all six strains. This investigation proved the occurence of VIM-2 β-lactamase among P. aeruginosa strains from Zagreb, Croatia. VIM-2 β -lactamase with similar properties was previously described in another region of Croatia, Italy, France, Spain, Greece, Taiwan and South Korea suggesting that this type of enzyme is widespread in Mediteranean region of Europe and in Far East. Key words: meropenem, imipenem, metallo-β-lactamases, resistance, Pseudomonas aeruginosa

Background: We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 14 mg/L) but high azithromycin MICs in absolute terms (1664 mg/L; n = 6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n = 1); and (3) high-level constitutive MLS (cMLS) resistance (n = 1). Here we analysed the genetic basis of these novel phenotypes. Methods: The presence of erm(A) and the absence of macrolide/lincosamide resistance genes erm(B), mef and cfr were confirmed by PCR. erm(A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm(A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (G) and putative configurations of the leader sequence were studied in silico. Results: Point mutations (G98A, A137C, C140T and G205A) were observed in the erm(A) regulatory region of all eight erm(A)-harbouring S. pyogenes. Five and two isolates belonged to emm77 and emm89 clones, respectively, and one isolate was an emm1. E. coli transformed with mutant erm(A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm(A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that G was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm(A) gene, explaining the cMLS phenotype 3. Conclusions: We report point mutations in the erm(A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm(A)-harbouring S. pyogenes.

A total of 78 isolates of Pseudomonas aeruginosa grouped according to the phenotype for ceftazidime and imipenem susceptibility/resistance were used to assess the accuracy of the Vitek 2 system in antimicrobial susceptibility testing. Comparisons were made with a MIC gradient test for piperacillin-tazobactam, ceftazidime, aztreonam, imipenem, meropenem, gentamicin, and ciprofloxacin. For the total of 546 isolate-antimicrobial combinations tested, the category agreement was 83.6%, with 2.0, 1.6, and 12.8% very major, major, and minor errors, respectively. Vitek 2 accuracy was influenced differently by the mechanism responsible for resistance, and interpretation of the results in relation to phenotype could improve the performance of the system.

Objectives: To test four 16-membered macrolides ( josamycin, spiramycin, midecamycin and rokitamycin) along with other compounds in the same class (erythromycin, clarithromycin, roxithromycin and azithromycin) plus clindamycin and telithromycin, against Streptococcus pyogenes and Streptococcus pneumoniae isolates with well-characterized resistance genotypes. Methods: Four hundred and eighty-six isolates of S. pyogenes and 375 isolates of S. pneumoniae were assayed for their macrolide susceptibilities and investigated by PCR to detect their different erythromycin resistance genes. All strains had been isolated over the period 2002–2003 from specimens of different human origin obtained in 14 different Italian centres. Results: All 16-membered macrolides showed very low MICs (MIC50s and MIC90s, � 0.06–0.5 mg/L) for the erythromycin-susceptible isolates and for those with the M phenotype, but the telithromycin MICs for the M-type isolates were at least four times higher (MIC90s, 0.5 mg/L). In S. pyogenes, the MIC50 so f 16-membered macrolides for the cMLSB isolates were � 256 mg/L, whereas that for telithromycin was 4 mg/L; the MIC50s of 16-membered macrolides and telithromycin ranged from � 0.06 to 0.5 mg/L for the iMLSB isolates with erm(A) and from 0.12 to � 256 mg/L for those with erm(B). In S. pneumoniae, the MIC50s of the 16-membered macrolides for the cMLSB isolates ranged from 0.5 to 128 mg/L, whereas for the iMLSB isolates their values ranged from � 0.06 to 4 mg/L; the MIC50s and MIC90s of telithromycin for both the cMLSB and the iMLSB isolates ranged from � 0.06 to 0.12 mg/L. Conclusions: MICs ranged for all the drugs, except telithromycin, from � 0.06 to � 256 mg/L, with 15% to 30% resistant S. pyogenes for all drugs tested except clindamycin (8%) and telithromycin (5.4%) and 10% to 40% resistant S. pneumoniae for all drugs tested except telithromycin (0.3%). In both S. pyogenes and S. pneumoniae, erythromycin resistance related to a mef gene meant that telithromycin MICs were definitely higher than in erythromycin-susceptible isolates, although telithromycin susceptibility was preserved in all cases. In S. pyogenes, the activity of both 16-membered macrolides and telithromycin against the iMLSB strains proved to be dependent on the erm gene involved, being greater against isolates with erm(A).

687 isolates of Streptococcus pyogenes and 600 isolates of Streptococcus pneumoniae , isolated over the period 2002-2003 from specimens of different human origin obtained in 16 different Italian centres, were assayed for their susceptibilities to different macrolides and to telithromycin, and were investigated by PCR to detect their different erythromycin resistance genes. 25.5% of the S. pyogenes isolates proved resistant to erythromycin, as well as to clarithromycin and azithromycin. 6.6% of the isolates proved non-susceptible to clindamycin. 4.9% of the isolates were non-susceptible to telithromycin. 22.3% of all erythromycin-resistant isolates exhibited cMLS B resistance, 50.3% iMLS B resistance, and 27.4% Mtype resistance. All cMLS B strains had the erm(B) gene, all M strains had the mef (A) gene, and no resistance genes were found in the erythromycin-susceptible strains. Roughly one quarter of the iMLS(B) strains had erm(A) and roughly three quarters erm(B). 35.2% of the S. pneumoniae isolates proved resistant to erythromycin, and virtually all of them also proved resistant to clarithromycin and azithromycin, too. Only 6.0% of the pneumococcal isolates were resistant to penicillin and a further 11.0% were intermediate. Only 0.2% of the isolates were nonsusceptible to telithromycin. 65.9% of all erythromycin-resistant S. pneumoniae isolates had cMLS B resistance, 18.0% had iMLS B resistance, and 16.1% had M-type resistance. All the MLS B-resistant isolates had an erm(B) gene, and all the M-type isolates had a mef gene. We conclude that macrolide resistance of streptococci still persists in Italy with incidences as high as 40%, more often than not being characterised by the MLS B phenotype. The ketolide telithromycin, structurally related to macrolides and most likely to substitute for them in a number of clinical uses, is confirmed as being extremely active even against recent clinical streptococcal isolates.

A multiply resistant strain of Salmonella enterica subsp. enterica serovar Virchow was isolated in November 2002 from a catheterized patient admitted to the SSK Training Hospital in Ankara, Turkey. This isolate showed an antimicrobial susceptibility pattern compatible with the presence of a CTX-M-type ESBL, namely resistance to cefotaxime, aztreonam and cefepime, and intermediate susceptibility to ceftazidime.On checking for the presence of the bla TEM bla SHV, and bla CTX-M resistance genes by PCR, negative results were obtained with the primers specific for SHV and TEM genes, while positive results were obtained with those specific for CTX-M-type genes. After sequencing, the beta-lactamase was identified as CTX-M-3.This is the first report of this enzyme in Salmonella Virchow and represents a further disquieting threat to the therapy of infections caused by Salmonella isolates.

We report on the first isolation of an extended-spectrum beta-lactamase-producing Leclercia adecarboxylata strain from the bloodstream in a 58-year-old man with acute myeloid leukemia. The strain, resistant to ceftazidime, cefotaxime, and aztreonam, produces the SHV-12 β-lactamase, one of the most common variants found in Italian nosocomial isolates of Enterobacteriaceae .

Immunoblotting with antibody against AcrA, an obligatory component of the AcrAB multidrug efflux system, showed that this protein was overexpressed by ≥170% in 9 of 10 clinical isolates of Esherichia coli with high-level ciprofloxacin resistance (MICs, ≥32 μg/ml) but not in any of the 15 isolates for which the MIC was ≤1 μg/ml.

A total of 8 Pseudomonas aeruginosa isolates was collected from 7 different patients in different wards of the University Hospital of Verona, Italy, from February 1997 to February 1998. The high level of resistance to carbapenems (imipenem minimum inhibitory concentration was always >128 microg/mL) and other broad-spectrum beta-lactams and the rate of imipenem hydrolysis and its inhibition by ethylenediamine-tetra-acetic acid were all suggestive of production of a carbapenem-hydrolyzing metallo-beta-lactamase. A specific DNA probe derived from the recently cloned bla(VIM-1) gene hybridized to all the isolates. A genomic DNA fingerprinting profile revealed clonal relatedness for 7 of 8 isolates. A description of this hospital outbreak is reported, the occurrence of which confirms that proliferation of metallo-beta-lactamase-producing strains multiply resistant to beta-lactams is already a reality outside Japan. These findings emphasize the need for early recognition of similar isolates.

To assess the spread of the new M phenotype, various erythromycin-resistant Streptococcus pyogenes strains from three Italian cities (Verona, Monza, Florence) were characterised. Each strain was analysed for the presence of genes ermAM and mefA, for the ability to accumulate radioactive erythromycin in the absence of sodium arsenate, for the protein T serological type, and for the DNA macrorestriction profile identified by means of pulsed-field gel electrophoresis. In a number of strains, the presence of the inducible ermAM gene was demonstrated; all these strains were negative in the efflux-pump detection assay, did not possess the mefA gene, and had similar restriction profiles. The strains with the efflux mechanism and mefA gene belonged to different serotypes. Of these, only one serotype, T4, was isolated in all three cities. The restriction profile analysis with SmaI and SfiI revealed a very close correlation between strains with the same serotype.

The in-vitro activity of MEN 10700, a novel penem, was compared with that of imipenem, ritipenem, ampicillin/sulbactam, cefotaxime, ciprofloxacin and amikacin against 1088 strains taken from 21 genera, including Gram-negative, Gram-positive and anaerobic bacteria. MIC data showed that MEN 10700 was very active against staphylococci and streptococci (MIC90 < or = 0.5 mg/L) and against most members of the Enterobacteriaceae (MIC90 < or = 2 mg/L), with reduced activity only against Providencia stuartii (MIC90 = 8 mg/L). MEN 10700 was also active against anaerobic species such as Clostridium perfringens and Bacteroides fragilis as well as Moraxella catarrhalis. It was moderately active against Enterococcus faecalis and inactive against Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Aeromonas spp. and Acinetobacter spp. Its antibacterial spectrum was thus slightly narrower than that of imipenem, but compared favourably with those of a third-generation cephalosporin and ritipenem. MEN 10700 was highly stable to a number of beta-lactamases and was a poor inducer of class I enzymes. It bound penicillin-binding protein 2 with the highest affinity and easily permeated the outer membrane of Escherichia coli.

A retrospective analysis of susceptibility data available for Group A streptococcal isolates collected between January 1990 and January 1996 at the Hospital Microbiology Laboratory of Monza (North Italy), showed a sharp rise in the erythromycin resistance rates during the last 3 years. Streptococcus pyogenes resistant to erythromycin accounted for approximately 1% of strains isolated between 1990 and 1992; the percentage then rose from 5% in 1993 to almost 39% in 1995. In January 1996, the resistance rates peaked to 81%. A prospective controlled study performed between March and May of 1996 to determine the percentage of erythromycin-resistant Group A streptococci isolated in Monza from untreated children with acute pharyngo-tonsillitis, gave further confirmation of a high rate of erythromycin resistance (47%) in this area. Molecular characterization by T-serotyping and pulse-field gel electrophoresis analysis of 25 erythromycin-resistant Group A streptococcal isolates, showed a relatively high degree of heterogeneity among these strains, demonstrating that the increased resistance is not caused by the spread of a single clone.

pneumoniae than the HEp-2 cells used in the present study, which may lead to lower endpoints. The discrepancy of MICs between these studies illustrates the problems of in vitro susceptibility testing of Chlamydia spp. in general and C. pneumoniae specifically. The methods are not standardized and the number of isolates available for testing are limited. Wide isolate-to-isolate variation in MICs has been seen with some antibiotics, most notably telithromycin. Data on the actual microbiological efficacy of quinolones for eradication of C. pneumoniae from patients with respiratory infection are limited; most treatment studies that have been published in the literature have used serology alone. The limited data that are available suggest that in vitro activity does not necessarily predict in vivo efficacy. Indeed, there are no data that demonstrate that an antibiotic with an MIC of 0.1 mg/L is more effective in eradication of C. pneumoniae than one with an MIC of 1 mg/L.

The prevalence of urinary tract infection increases substantially with advancing age, and in institutionalized elderly subjects it increases with functional disability and length of residence (16). Bacteria of the genus Providencia, with special reference to Providencia stuartii, are among those most frequently isolated from urine specimens from elderly subjects, and they are gaining ground as prominent organisms in bacteriuria among both institutionalized patients (16, 20, 21) and patients receiving home care (7, 8, 20). The ability of this species to persist in the catheterized urinary tract seems to be