Human Staphylococcus aureus lineages among Zoological Park residents in Greece.

Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL) and tst (toxic shock syndrome toxin-1) were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST), spa type and Pulsed-Field Gel Electrophoresis (PFGE). Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst-negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred.

A 12-year survey of methicillin-resistant Staphylococcus aureus infections in Greece: ST80-IV epidemic?

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) infections. Severe MRSA infections have been associated with the virulence factor Panton-Valentine leukocidin (PVL). The aim of this study was to investigate susceptibility patterns, the presence of toxin genes, including that encoding PVL, and clonality among MRSA isolates collected from patients in Greece over a 12-year period. MRSA isolates were collected from January 2001 to December 2012 from six different hospitals. Antibiotic susceptibility was determined with the disk diffusion method and the Etest. The presence of the toxic shock syndrome toxin-1 gene (tst), the enterotoxin gene cluster (egc) and the PVL gene was tested with PCR. The genotypic characteristics of the strains were analysed by SCCmec and agr typing, and clonality was determined with pulsed-field gel electrophoresis and multilocus sequence typing. An increasing rate of MRSA among S. aureus infections was detected up to 2008. The majority of PVL-positive MRSA isolates belonged to a single clone, sequence type (ST)80-IV, which was disseminated both in the community and in hospitals, especially during the warmest months of the year. Carriage of tst was associated with ST30-IV, whereas egc was distributed in different clones. CA-MRSA isolates were recovered mainly from skin and soft tissue infections, whereas HA-MRSA isolates were associated with surgical and wound infections. During the period 2001-2012, ST80-IV predominated in the community and infiltrated the hospital settings in Greece, successfully replacing other PVL-positive clones. The predominance of ST239-III in HA-MRSA infections was constant, whereas new clones have also emerged. Polyclonality was statistically significantly higher among CA-MRSA isolates and isolates from adult patients.

Mycobacterium kansasii cutaneous infection in a patient with sarcoidosis treated with anti-TNF agents.

We describe a Mycobacterium kansasii cutaneous infection that was diagnosed in a 52-year-old female patient with sarcoidosis receiving anti-TNF agents. The diagnosis was based on the positive culture of the foot ulcerative tissue. The isolation and identification of bacterium was based on phenotypic and molecular methods. Therapy and follow-up of the patient is discussed.

Modified directly observed treatment for tuberculosis versus self-administered therapy: an observational study in rural Greece.

INTRODUCTION: Directly Observed Treatment (DOT) is the key element of DOTS (directly observed treatment, short course), part of the internationally recommended control strategy for tuberculosis (TB). The evaluation of DOT has not been widely evaluated in rural areas in developed settings. The aim of this pilot study was to evaluate a modified DOT program (MDOT) by a general practitioner (GP) in a rural area of southwest Greece, where there is substantial underreporting of TB cases. METHODS: Thirteen new TB cases with 30 close contacts were compared with 41 past-treated TB subjects (controls) with 111 close contacts in this observational, case-control study. Home visits by a GP were conducted and comparison of various data (laboratory findings, treatment outcomes, questionnaire-based parameters, on-site recorded conditions) was performed in both newly detected pulmonary TB cases and previously treated TB cases managed without DOT intervention. RESULTS: MDOT by GP implementation revealed that 11 cases (84.6%) were successfully treated, one (7.7%) case died, and one (7.7%) was lost to follow up. None of the close contacts of new TB cases was infected with active TB, while 6.3% of previously-treated TB subjects were infected with active TB and had to receive a complete anti-TB regimen. Chemoprophylaxis was administered to 13.3% of close contacts of new cases; whereas 12.6% of close contacts of previously-treated patients received chemoprophylaxis. CONCLUSION: This pilot study revealed that a GP is able to implement a program based on DOT resulting in high treatment adherence and prevention of TB compared with the conventional self-administration of treatment.

The combined effect of surface chemistry and flow conditions on Staphylococcus epidermidis adhesion and ica operon expression.

The assessment of biomaterial susceptibility to infection relies mainly on the analysis of macroscopic bacterial responses to material interactions, usually under static conditions. However, new technologies permit a more profound understanding of the molecular basis of bacteria-biomaterial interactions. In this study, we combine both conventional phenotypic analysis - using confocal microscopy - and genotypic analysis - using the relative reverse transcription polymerase chain reaction (RT-PCR) - to examine the interaction of bacteria with OH- and CH3-terminated glass surfaces, under dynamic flow conditions. Bacterial adhesion, as well as slime production and biofilm formation, was much higher on the CH3-terminated than on the OH-terminated glass - for four Staphylococcus epidermidis strains. This was in agreement with the icaA and icaD gene expression results that showed increased expression for the bacteria adhering to the CH3-terminated substrate, especially under the higher shear rate. Therefore, the combined effect of the surface chemistry and shear significantly influence the adhesion and phenotype of interacting bacterial cells, while there are putative links between phenotypic responses to bacteria-material interactions and gene-expression profile alterations. This indicates that analysis of gene expression not only can greatly refine our knowledge of bacteria-material interactions, but also yield novel biomarkers for potential use in biocompatibility assessment.

The first case of Staphylococcus aureus ST398 causing bacteremia in an immunocompromised patient in Greece.

We describe a case of catheter-related bloodstream infection, in a patient with colon cancer, caused by a methicillin-sensitive Staphylococcus aureus strain, nontypeable by pulsed field gel electrophoresis of SmaI macrorestriction fragment analysis, belonging to ST398. The patient recovered after daptomycin therapy. This is the first report that documents the emergence of ST398 in Greece.

Clinical and microbiological profile of persistent coagulase-negative staphylococcal bacteraemia in neonates.

An atypical pattern of coagulase-negative staphylococcal (CoNS) sepsis, characterized by persistence despite aggressive antibiotic therapy, has been described in neonates cared for in neonatal intensive-care units. Our aim was to analyse the clinical, microbiological and molecular determinants of this persistent CoNS bacteraemia. Neonates with late-onset CoNS bacteraemia were studied for a 2-year period. Demographic, clinical, laboratory, microbiological and molecular data were compared between neonates with persistent (≥3 consecutive positive blood cultures) and non-persistent CoNS bacteraemia. Twenty-nine infants with persistent and 43 with non-persistent bacteraemia were identified, with no significant differences regarding demographic and clinical characteristics between the two groups. Of a total of 170 CoNS isolates, 80 showed biofilm production (54 persistent and 26 non-persistent; p 0.013), whereas 127 were positive for the icaA and icaD genes (74 persistent and 53 non-persistent; p 0.598). Sixty ica-positive isolates did not produce slime, whereas 13 ica-negative isolates showed biofilm production. Endotracheal intubation and the presence of central vascular catheters were significant risk factors for persistent bacteraemia, but, in a logistic regression model, only biofilm production was significantly related to the persistent form of the disease (p 0.005). In this study, persistent CoNS sepsis in neonates requiring intensive care was not related to most of the known clinical risk factors, and it was associated with severe thrombocytopenia. Isolates associated with persistent bacteraemia were more likely to produce biofilm, independently of the presence of the ica operon.

Absolute and relative real-time PCR in the quantification of tst gene expression among methicillin-resistant Staphylococcus aureus: evaluation by two mathematical models.

AIM: Absolute and relative quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) by the use of two mathematical models were applied in order to study the expression of tst gene encoding the toxic shock syndrome toxin-1 (TSST-1), among methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Thirteen epidemic MRSA belonging to different clones and carrying a variety of toxin genes were selected. tst gene expression was achieved by using absolute and relative quantitative real-time RT-PCR and the SYBR Green I. Absolute RT-PCR showed a statistically significant higher level of tst expression among strains isolated from soft tissue infections. Relative quantification was performed in relation to 23S rRNA expression by the application of two mathematical models, the 2(-DeltaDeltaCt) and the Pfaffl analysis methods. CONCLUSIONS: tst gene expression was best calculated by the relative real-time RT-PCR analysis applying the Pfaffl analysis method, taking into account the reactions' efficiencies. Level of tst expression was related to patients' infection and did not depend on the MRSA genetic profile. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the application of the Pfaffl analysis method in the evaluation of relative real-time RT-PCR is more adequate.

Clonality of slime-producing methicillin-resistant coagulase-negative staphylococci disseminated in the neonatal intensive care unit of a university hospital.

Methicillin-resistant coagulase-negative staphylococci (MR-CNS) (n = 132), isolated from pre-term neonates, were analysed to determine their antibiotic resistance patterns, clonal distribution, biofilm production and the presence of the ica operon. All MR-CNS were multiresistant, and 89% produced slime. A major clone was identified (77 isolates) among 115 Staphylococcus epidermidis isolates. Ten of 16 Staphylococcus haemolyticus isolates also belonged to a single clone. Most (80%) slime-positive isolates possessed all the ica genes tested, while the remaining 23 (20%) had a variety of gene combinations. The entire ica cluster was detected in three of 15 slime-negative isolates. One major and two minor slime-positive, multiresistant MR-CNS clones had disseminated among hospitalised pre-term neonates.

Comparison of two commercial methods with PCR restriction fragment length polymorphism of the tuf gene in the identification of coagulase-negative staphylococci.

AIMS: Two commercial methods for the identification of coagulase-negative staphylococci (CNS) were compared with the restriction fragment length polymorphism (RFLP) of the amplified tuf gene, which served as the reference method. METHODS AND RESULTS: One hundred and forty-five CNS were evaluated using the API 32 Staph ID and the Crystal GP/ID BBL systems. The PCR-RFLP of the tuf gene served as the reference method. The APIStaph and the GP/ID BBL had an overall rate of agreement with the molecular method of 58.6% and 46.2% respectively, with the inability of the GP/ID BBL to characterize 11.7% of the isolates. The APIStaph showed higher sensitivity and better agreement than the GP/ID BBL with the PCR-RFLP, except for Staphylococcus hominis and Staphylococcus capitis. CONCLUSIONS: Neither of the commercial systems was as reliable as the PCR-RFLP method for identifying isolates of CNS. Overall the APIStaph had better agreement with the PCR-RFLP than the GP/ID system. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that the PCR-RFLP method is more reliable than the two commercial systems tested, suggesting that it is more reliable for routinely identifying CNS.

Spread of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin genes during a 3-year period in Greece.

Three collections of Staphylococcus aureus isolates (n = 1,058) were investigated to assess the spread of Panton-Valentine leukocidin (PVL)-producing strains in Greece and their association with skin and soft-tissue infections (SSTIs). The isolates were collected during 2001-2003 from inpatients and outpatients with invasive infections in two distinct geographical areas. Clonal types were identified according to their ClaI-mecA::ClaI-Tn554::pulsed-field gel electrophoresis patterns, and the presence of the lukS-PV and lukF-PV genes was assessed by PCR. In total, 287 (27%) S. aureus isolates carried the PVL genes: 45% of methicillin-resistant S. aureus (MRSA) and 12% of methicillin-sensitive S. aureus (MSSA). All the PVL-positive MRSA isolates belonged to a single clone that was disseminated in the community and hospitals. The PVL-positive MSSA isolates were polyclonal, with 14 of 65 isolates being associated with hospital-acquired infections. The community-acquired isolates were from SSTIs, while the hospital-acquired isolates were associated with surgical wound infections, especially those involving prosthetic devices. Thus, a unique clone of PVL-positive MRSA has spread in both the community and the hospital setting in Greece, and has replaced older clonal types.