Comparison of the Staphylococcus QuickFISH BC test with the tube coagulase test performed on positive blood cultures for evaluation and application in a clinical routine setting.

Many studies demonstrate that delayed proper therapy in bloodstream infections caused by Staphylococcus aureus increases the mortality rate, emphasizing the need to shorten the turnaround time for positive blood cultures. Different techniques are currently available, from phenotypic methods to more complex tests such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), real-time PCR (RT-PCR), and fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH). This study evaluated the performance of the Staphylococcus QuickFISH BC test (QFT), a novel FISH methodology, compared with the direct tube coagulase test (DTCT) on blood cultures exhibiting Gram-positive cocci in clusters. A total of 173 blood cultures collected from 128 different patients were analyzed using the DTCT, evaluated after both 4 and 24 h, and the QFT. A total of 179 isolates were identified using the Vitek2 system. Thirty-five out of 35 Staphylococcus aureus were correctly identified by the QFT (sensitivity = 100%), with a specificity of 100% (no green fluorescence was detected for strains different from S. aureus). The DTCT was positive after 4 h for 28 out of the 35 samples (sensitivity = 80%) and after 24 h for 31 out of the 35 samples (sensitivity = 88.57%). Among the remaining 144 isolates, one was then identified as Corynebacterium striatum and two as Micrococcus luteus. QFT identified 139 out of the 141 coagulase-negative staphylococci (CoNS) (sensitivity = 98.58%), showing again a specificity of 100% (no fluorescent red signals were detected for strains different from CoNS). We also discuss also the implementation process of this methodology in our setting, with particular emphasis on the workflow and the cost-effectiveness.

Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit.

Following the identification of two clinical isolates of vancomycin-resistant enterococci (VRE) from intensive care unit (ICU) patients, a surveillance programme detected that six of eight ICU patients were colonised by VRE. Standard epidemic control measures were instituted in the ICU. During a 16-month period, 13 (2.5%) of 509 ICU patients had VRE-positive swabs upon admission, and 43 (8.7%) of 496 VRE-negative patients were colonised by VRE in the ICU. Patients who acquired VRE in the ICU had a longer ICU stay (p < 0.0001). No other statistically significant differences were demonstrated. Two patients had documented infection (infection/colonisation index, 3.6%; overall VRE infection frequency, 0.4%), but both recovered and were discharged. VRE colonisation did not increase the mortality rate. Automated ribotyping identified three clusters containing, respectively, the first 52 Enterococcus faecium isolates, two Enterococcus faecalis isolates, and two further isolates of E. faecium. Multilocus sequence typing demonstrated that two E. faecium isolates representative of the two ribotypes belonged to sequence types 78 and 18, and that these two isolates belonged to the epidemic lineage C1, which includes isolates with a wide circulation in northern Italy. The outbreak was controlled by continuous implementation of the infection control programme, and by the opening of a new unit with an improved structural design and hand-washing facilities.

Identification of coagulase-negative staphylococci other than Staphylococcus epidermidis by automated ribotyping.

As routine identification of coagulase-negative staphylococci is problematic, the performance of automated ribotyping was evaluated for identification of coagulase-negative staphylococci other than Staphylococcus epidermidis. In total, 177 isolates were tested, comprising 149 isolates from blood samples, 15 isolates that were not identified by internal transcribed spacer (ITS)-PCR in a previous study, and 13 reference strains. The identification results were compared with those obtained by the API 20 Staph system, with standard phenotypic and molecular methods as reference. Most (n = 166; 93.8%) isolates were identified correctly by automated ribotyping. For 61 isolates, API 20 Staph and ribotyping were in agreement, but for 105 isolates, ribotyping provided correct identification and API 20 Staph did not. Four isolates not identified by automated ribotyping were recognised correctly by API 20 Staph. The remaining seven isolates could not be identified by either of the two methods. Automated ribotyping was able to distinguish Staphylococcus capitis reliably from Staphylococcus caprae. The results demonstrate the value of automated ribotyping for identification of coagulase-negative Staphylococcus (CoNS) isolates from human sources and may help to clarify the clinical relevance of CoNS species. In addition, automated ribotyping was able to detect polymorphisms that may be useful for epidemiological purposes within S. capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus simulans, S. caprae, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus pasteuri and Staphylococcus xylosus.

Evaluation of carbapenem-resistant Enterobacteriaceae in an Italian setting: report from the trench.

The spread of carbapenem resistant Enterobacteriaceae (CRE) has recently become a matter of concern in public health, mainly due to the wide distribution of carbapenemase genes. Italy is a country considered endemic for the spread of blaKPC Klebsiella pneumoniae (KP). The aim of this study was to depict the epidemiological trend of CRE in one Italian hospital over a long period (3 years surveillance, from May 2011 to April 2014). Based on defined MIC cut-off for specific carbapenems, 164 strains isolated from 146 different patients were analyzed both phenotypically and genotypically to establish the resistance genes. Molecular typing was performed using the RAPD technique. 77 strains were demonstrated to harbor the blaKPC gene (73 KP, 4 Escherichia coli - EC), 51 strains the blaVIM gene (44 KP, 3 EC, 2 Enterobacter cloacae and 2 Klebsiella oxytoca), 8 the blaNDM gene (3 KP, 4 EC and one Providencia stuartii), 3 the blaOXA-48 gene (2 KP, 1 EC), whereas 25 out of the 164 isolates (of different genera and species) had a negative multiplex-PCR amplification for all the targets tested. 39 out of the 164 strains analyzed (23.8%) revealed discrepancies between the MICs obtained with automated instrument and gradient MICs of more than two logs of difference; the broth microdilution provided a better agreement with the results obtained with the gradient MIC. The use of RAPD allowed to distinguish different clusters, closely related, both for blaKPC and for blaVIM KP.

Rapid drug susceptibility of Mycobacterium avium complex using a fluorescence quenching method.

Mycobacteria Growth Indicator Tube (MGIT) is a recently introduced rapid growth detection method which uses an oxygen quenched fluorescent indicator. The present study evaluated the ability of this new method to determine the drug susceptibility of Mycobacterium avium complex (MAC). Thirty strains recovered from patients with AIDS were tested for susceptibility to clarithromycin, rifabutin, ciprofloxacin, azithromycin and amikacin using MGIT. Results were compared to susceptibilities determined by the agar dilution method. The results obtained showed a 100% correlation between MGIT and the agar dilution method for rifabutin and clarithromycin. There was a 100% correlation between the two methods for azithromycin against 27 strains. MGIT was well correlated with the agar dilution method for detecting resistance to clarithromycin, rifabutin and azithromycin in 4 days, but the correlation was poor when susceptibilities to ciprofloxacin and amikacin were determined. This rapid method is non-radiometric, noninvasive and does not require any special instruments.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in an Italian intensive care unit: clinical and therapeutical remarks.

In this study we evaluated the prevalence of Enterobacteriaceae and the epidemiology of ESBL+ microorganisms in an ICU of our Institution over a 5-year period and analyzed the clinical features and outcomes of the infections caused by these microorganisms. The most frequent ESBL+ isolate was Proteus mirabilis (69 isolates, 58%); a high rate of positive results in the double-disk synergy test (DDS) was also recognized for Klebsiella pneumoniae (52 isolates, 51%), whereas this phenomenon was observed less frequently in other species. In 312 cases the isolated microorganism was considered to be the cause of infection; we documented 103 wound infections, 89 UTIs, 62 LRTIs, 30 primary bacteremias, 27 infections of indwelling catheters and 1 CNS infection. The overall mortality rate due to ESBL+ strains was 1%, compared with 10.6% rate caused by ESBL-negative Enterobacteriaceae. This could be explained because ESBL+ strains caused mostly localized infections (wound infections and UTIs), whereas systemic or severe infections were sustained by ESBL-negative strains, and therapy with carbapenems was started promptly after ESBL+ isolation (always within 24h after strain isolation).

Rapid identification of Mycobacterium tuberculosis and Mycobacterium avium by polymerase chain reaction and restriction enzyme analysis within sigma factor regions.

A single Polymerase Chain Reaction (PCR) within the rpoV gene was developed to rapidly distinguish mycobacteria isolated from clinical specimens. The species identifications of Mycobacterium avium complex (MAC) and Mycobacterium tuberculosis were congruent with standard typing techniques. The analysis was targeted toward the identification of species-specific markers for the clinically relevant M. tuberculosis and M. avium. In addition, HaeIII digestion of the amplification products yielded isolates-specific bands.

Typing of methicillin-resistant Staphylococcus aureus (MRSA) strains from an intensive care unit by random amplified polymorphic DNA (RAPD).

The identification and control of methicillin-resistant Staphylococcus aureus (MRSA) is of primary concern in intensive care units (ICUs) worldwide. The introduction and circulation of particular strains is best studied by genomic procedures and random amplified polymorphic DNA (RAPD) is well suited for this task. In this study 14 isolates of MRSA, obtained over an 8 month period from the blood cultures of 12 patients in an ICU at our hospital, were typed by RAPD method using seven primers. Three separate groups were distinguished and clustering of certain types in time and space was noted. These results suggest that although different strains of MRSA were involved in this outbreak, cross-infection with individual types occurred. RAPD fingerprinting is a relatively simple method that allows epidemiologic investigation of MRSA outbreaks in hospital infection.

[Methicillin-resistant Staphylococcus aureus (MRSA) in an intensive care unit: a one-year survey]. / Colonizzazioni e infezioni da Staphylococcus aureus meticillino-resistente (SAMR) in una terapia intensiva polivalente: analisi su un anno di osservazione.

Methicillin-resistant Staphylococcus aureus (MRSA) is frequently isolated in nosocomial outbreaks. In our study, we analysed the occurrence of colonisation and infection in an Intensive Care Unit of our hospital during a 12-month period. We also evaluated the possibility of using automated ribotyping as a molecular method in order to type the isolates. Twice a week a nasal swab and a rectal swab were performed on all patients; from ventilator-assisted patients, a sputum culture was also taken. All the MRSA isolated were identified by using commonly phenotypic procedures and on all isolates susceptibility tests were performed. An automated ribotyping using EcoRI was also done. Out of 292 patients enrolled in the study, 205 were never colonised (group N); among the other 87 who were colonised by MRSA (29.8%), 40 patients (group A) were MRSA carriers at the time of admission, while 47 (group B) were colonised in the ICU. Twenty-seven patients (11 from group A, 15 from group B and 1 from group N) developed 31 infections due to MRSA. Patients from group A exhibited, as a rule, worse clinical conditions than those from the other two groups. For the former group, MRSA infection was frequently systemic (sepsis), while in group B pneumonia was the predominant infection. The prevalence of colonisations in our study was 30%, which is a value comparable to those presented by other authors in similar cases. MRSA colonisation is a necessary condition for subsequent infections in almost all cases, with an average lag of 7 days. Susceptibility tests were non-discriminating among the isolates: all the strains were susceptible to glycopeptides; nearly all of them were resistant to erythromycin, clindamycin, ciprofloxacin and gentamicin. Automated ribotyping allowed us to distinguish 12 different ribogroups, the most frequent of which was composed of 146 isolates. In our study, this molecular method was able to define a possible endemic clone that should be better investigated by using methods with a higher discriminatory power, such as RAPD or PFGE. The method that we employed is highly reliable, easy to perform and not time-consuming. In our opinion, it could be the method of choice in the first screening of high numbers of isolates.

Widespread carbapenem resistant Acinetobacter baumannii clones in Italian hospitals revealed by a multicenter study.

Population diversity, susceptibility to antibiotics including carbapenems of 277 Acinetobacter baumannii strains collected in 17 Italian hospitals over a 6-months' period was assessed. Semi-automated rep-PCR was used for screening strains for genotypic relatedness. AFLP analysis and MLST were used as definitive methods for strain, species and/or clone identification. Among the 277 strains, 49 rep-PCR types were distinguished with four types (1-4) predominant, indicating both intra- and interhospital spread. AFLP analysis allowed to distinguish 51 types and largely confirmed rep-typing results. Isolates with predominant rep-types 1 and 2 (in 3 and 9 hospitals) were allocated to EU clones I and II, respectively. Rep-type 3 (8 hospitals) belonged to a new clone ("Italian clone"). Rep-type 4 was found in 2 neighbouring hospitals. Two isolates from 2 locations belonged to EU clone III. Twenty-five isolates were identified by AFLP-analysis to A. pittii, emphasizing misidentification by phenotypic methods. MLST confirmed clone identification by AFLP; demonstrating also that the "Italian clone" was ST78, recently detected in different Mediterranean countries. Multidrug resistance, defined as resistance to 9 out of the 11 drugs tested, was common in 10 out of 17 hospitals. The high prevalence of carbapenem resistance was associated with OXA-58 found in 9 out of the 10 hospitals. A high percentage of noted very major errors in susceptibility testing, especially for amikacin and meropenem, was probably due to heteroresistant strains. The occurrence of carbapenem and multidrug resistance in A. baumannii was mainly confined to a limited number of clonal lineages of A. baumannii.

Bacillus cereus fatal bacteremia and apparent association with nosocomial transmission in an intensive care unit.

Bacillus cereus has sometimes been implicated in food poisoning and in opportunistic infections of seriously ill patients. This report describes an unusual case of persistent bacteremia and multiple organ failure associated with B. cereus in a patient admitted to our institution for lung cancer. The patient was undergoing treatment with an antimicrobial agent (imipenem) that was shown to be effective against the micro-organism in vitro. No portal of entry for the strain was detected. After treatment with vancomycin, also shown to be effective in vitro, no clinical improvement was noted and the patient died. Molecular studies showed that the same strain caused an episode of pseudobacteremia in another patient admitted to the same ICU room.

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: phylogenetic implications.

The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%-49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.

Vancomycin-resistant Enterococcus faecium infection in three children given allogeneic hematopoietic stem cell transplantation: clinical and microbiologic features.

BACKGROUND AND OBJECTIVES: The emergence of vancomycin-resistant enterococci (VRE) as nosocomial pathogens is a major problem in the US; in Europe, VRE nosocomial infections are uncommon and only rarely have been reported in Pediatric or Neonatal Units. The aim of this study is to report on the clinical and microbiological features of VRE infections in 3 children given hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS: Five episodes of vancomycin-resistant Enterococcus faecium (VREF) infection were diagnosed in 3 children given an allogeneic HSCT. Molecular methods, such as random amplification of polymorphic DNA (RAPD) fingerprinting and automated ribotyping, were used in order to define the circulation of strains. RESULTS: All the isolates were resistant to all commercially available agents and showed the VanA genotypic profile. All children were successfully treated with the combination of quinupristin/dalfopristin (QD) plus teicoplanin (TEC), although treatment was not sufficient to eradicate the micro-organism promptly from the gastrointestinal tract. All our children are still alive. After the first isolation of VRE, a surveillance protocol was started and we documented that the rate of colonization in children and their mothers was less than 1.5%. The RAPD method demonstrated the possible nosocomial transmission of one strain. INTERPRETATION AND CONCLUSIONS: Our experience demonstrates that VRE infection is a life-threatening complication in children given HSCT. Prompt diagnosis of this infection and its treatment with the combination of QD and TEC can successfully manage this severe infection in profoundly immunocompromised patients.

Catheter-related bacteremia due to Lactobacillus rhamnosus in a single-lung transplant recipient.

There is still a major debate about the pathogenicity of Lactobacillus spp. and some reports emphasize that these microorganisms are never isolated from endovascular devices. In this report we present a case of catheter-related bacteremia due to L. rhamnosus in a patient who underwent a single-lung transplant.

Catheter-related bacteremia due to Kocuria kristinae in a patient with ovarian cancer.

We report on the first case of a catheter-related recurrent bacteremia caused by Kocuria kristinae, a gram-positive microorganism belonging to the family Micrococcaceae, in a 51-year-old woman with ovarian cancer. This unusual pathogen may cause opportunistic infections in patients with severe underlying diseases.