Extended-spectrum beta-lactamases and plasmid diversity in urinary isolates of Escherichia coli in Croatia: a nation-wide, multicentric, retrospective study.

In recent years, a dramatic increase in the prevalence of Escherichia coli strains producing extended-spectrum ß-lactamases (ESBLs) has been observed - both in the community and in healthcare settings. This multicentric study aimed to characterize ESBLs produced by E. coli isolates causing hospital-onset and community urinary tract infections, as well as to compare their antimicrobial sensitivity patterns, ß-lactamase content and plasmid types. Phenotypic tests for the detection of ESBLs and plasmid-mediated AmpC ß-lactamases were initially pursued, followed by molecular detection of resistance genes, plasmid characterization, genotyping with pulsed-field gel electrophoresis and whole genome sequencing (WGS). The isolates exhibited high level of resistance to expanded-spectrum cephalosporins (ESC) and carried CTX-M (cefotaximase-Munich) or TEM (Temoniera) ß-lactamases. All six representative isolates subjected to WGS belonged to the widespread clone ST131. In conclusion, our study demonstrated dissemination of group 1 CTX-M positive E. coli in different geographic regions of Croatia, but also different components of the health care systems (hospitals, nursing homes and the community) and confirmed the switch from SHV-2 (suphydril variant) and SHV-5 ESBLs to the nation-wide predominance of group 1 CTX-M ß-lactamases. Different plasmids were shown to be associated with the dissemination of blaCTX-M genes in different geographic regions of Croatia.

Hidden Carbapenem Resistance in OXA-48 and Extended-Spectrum ß-Lactamase-Positive Escherichia coli.

The purpose of this study was to report the identification OXA-48 carbapenemase in seven extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli clinical isolates, fully susceptible to all carbapenems by disk diffusion and E-test methods, but with borderline minimal inhibitory concentration (MIC) values of ertapenem. This report points to the necessity for determination of carbapenem MICs in ESBL-positive E. coli isolates and additional phenotypic testing for carbapenemases in all isolates with borderline ertapenem MIC defined by EUCAST. The isolates showed a high level of resistance to expanded-spectrum cephalosporins because of the production of an additional ESBL belonging to CTX-M family. All isolates and their respective tranconjugants were found to possess L plasmid. Pulsed-field gel electrophoresis analysis revealed two clusters containing highly related isolates. The global spread of multidrug-resistant E. coli should be monitored closely because of the ability of isolates to rapidly obtain additional antibiotic resistance traits such as plasmid-mediated OXA-48 genes.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PURPOSE: A dramatic increase in OXA-48 ß-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. METHODS: Carbapenemase and other ß-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 ß-lactamases. In addition to the ß-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, ß-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Leuconostoc sp. Meningitis in a Patient Treated with Rituximab for Mantle Cell Lymphoma.

We present a 64-year-old man who was treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemoimmunotherapy for mantle cell lymphoma and developed purulent meningitis, probably caused by Leuconostoc sp. The patient had severe hypogammaglobulinemia, which is a possible complication of rituximab therapy. To our knowledge and after reviewing the available medical literature, this is the first described case of purulent meningitis caused by Leuconostoc sp. in a patient with mantle cell lymphoma that appeared after treatment with the R-CHOP protocol. The diagnosis of purulent meningitis was based on clinical, laboratory and cytological cerebrospinal fluid findings, in addition to blood culture results in which we isolated Leuconostoc sp. The patient was treated with meropenem with full recovery.

Bacterial colonization or infection in chronic sinusitis.

The aim of this study was the determination of bacteria present in maxillary and ethmoid cavities in patients with chronic sinusitis and to correlate these findings with bacteria simultaneously present in their nasopharynx. The purpose of this correlation was to establish the role of bacteria found in chronically inflamed sinuses and to evaluate if the bacteria present colonized or infected sinus mucosa. Nasopharyngeal and sinus swabs of 65 patients that underwent functional endoscopic sinus surgery were cultivated and at the same time the presence of leukocytes were determined in each swab. The most frequently found bacteria in nasopharynx were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus spp., Streptococcus viridans and Streptococcus pneumoniae. Maxillary or ethmoidal sinus swabs yielded bacterial growth in 47 (72.31%) patients. The most frequently found bacteria in sinuses were Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella spp. and Streptococci (pneumoniae, viridans and spp.). The insignificant number of leukocytes was present in each sinus and nasopharyngeal swab. Every published microbiology study of chronic sinusitis proved that sinus mucosa were colonized with bacteria and not infected, yet antibiotic therapy was discussed making no difference between infection and colonization. Chronic sinusitis should be considered a chronic inflammatory condition rather than bacterial infection, so routine antibiotic therapy should be avoided. Empiric antibiotic therapy should be prescribed only in cases when the acute exacerbation of chronic sinusitis occurs and the antibiotics prescribed should aim the usual bacteria causing acute sinusitis. In case of therapy failure, antibiotics should be changed having in mind that under certain circumstances any bacteria colonizing sinus mucosa can cause acute exacerbation of chronic sinusitis.

Scopulariopsis brevicaulis as the cause of dermatomycosis.

Scopulariopsis (S.) brevicaulis (Saccardo) Bainier 1907 is a ubiquitous fungus frequently isolated as a saprophyte from various layers of the soil, wood, straw, paper, food, and occasionally in animals and humans. This nondermatophyte filamentous fungus is multiresistant and is frequently associated with onychomycosis in humans. In the last two decades, the number of reports on its pathogenic role in different localized and disseminated infections has been on an increase. Identification was done by native KOH microscopy and culture on the Mycobios selective agar (Biolife). From January 1, 2002 till January 23, 2008, a total of 7161 samples were examined by use of mycology methods; S. brevicaulis accounted for 39 (2.2%) of 1834 (25.6%) positive samples. During the study period, S. brevicaulis was isolated from nail, skin and scalp scrapings of 39 patients (17 male and 22 female), mean age ( SD) 43.920.7 (range 8-87) years. Specimens were most frequently obtained from the foot (n=16; 41%), i.e. great toe nail in 10 and other foot areas in 6 cases, followed by palm and fingers in 8 (20.5%), scalp in 3 (7.7%) and other parts of the body in 12 (30.8%) cases. Most of the study subjects lived in a rural setting (n=22; 56%), working as farmers in close contact with the soil and domestic animals. Seventeen (44%) subjects were from urban setting (n=9) or unknown place of residence (n=8). Underlying risk factors were present in 29 (74%) of 39 study subjects, some of them with multiple risk factors. Besides close contact with the soil, the most common predisposing factors were various dermatoses (atopic dermatitis, psoriasis, dysseborrhea, etc.), lower extremity circulatory insufficiency, trauma, microtrauma, and metabolic disorders. Although the clinical picture of onychomycosis caused by S. brevicaulis shows some specific features, timely sampling for mycology is crucial to verify the diagnosis and to identify the causative agent prior to the introduction of appropriate therapy for dermatomycosis.

[The importance of Streptococcus bovis systemic infections]. / Vaznost sistemskih infekcija izazvanih bakterijom Streptococcus bovis.

Streptococcus bovis is relatively common cause of bacteremia and endocarditis, especially in older persons, or in patients that have some kind of chronic disease. These infections are frequently connected with malignant, potentially malignant, or benign colorectal neoplasia. Hematogenous dissemination of Streptococcus bovis could result with various clinical manifestations, namely purulent meningitis, brain abscess, osteomyelitis, spondylodiscitis, and many different kinds of infections in AIDS patients are reported. In this report, two patients hospitalized at the Departement of Infectious Diseases at the General Hospital "Dr. Josip Bencevic" in Slavonski Brod are presented. The first patient was addmited to the hospital because of fever of unknown origin, and in his blood cultures, Streptococcus bovis was isolated. He was an older man, who had undergone prostatectomy due prostatic adenoma several years before. The other patient, previously completely healthy younger man, suffered purulent meningitis caused by the same microorganism. Colon endoscopy was performed in both patients and it revealed colon polyps. Histologically, in both cases, those were benign neoplasia. In Croatia, until this report, there have been no other reports about patients suffering systemic Streptococcus bovis infections. At the same time, this report describes Streptococcus bovis purulent meningitis in a previously healthy adult, which is also extremely rare in the medical literature. Since Streptococcus bovis infections are associated with colon carcinoma, it is imperative to perform colonoscopy in each patient suffering infection with this germ, and to consider him as a high risk patient for developing colon cancer.

[Is the methicillin-resistant Staphylococcus aureus heteroresistant to vancomycin in Croatia?]. / Ima li u Hrvatskoj sojeva Staphylococcus aureus heterorezistentnih na vankomicin?

INTRODUCTION: Staphylococcus (S.) aureus with reduced susceptibility to vancomycin has attracted much attention all over the world since the first report of Staphylococcus aureus isolate intermediarily resistant to vancomycin (VISA) in Japan 1997. Other authors from different parts of the world have also described VISA isolates in patients with treatment failures after prolonged vancomycin therapy. Most of the isolates were heterogeneously resistant (hVISA), i. e. only a part of the population showed resistance and the rest showed susceptibility to vancomycin. AIM: Aim of the study was to determine the existence of methicillin-resistant S. aureus (MRSA) strains with reduced susceptibility to vancomycin in Croatia. METHODS: Abbreviated population analysis was used for detection of strains with reduced susceptibility to vancomycin. Forty-eight MRSA strains from three different hospitals in Croatia were tested on brain-heart infusion agar (BHIA) screen plate containing 4 mg/L vancomycin. Thirty-three (68.7%) strains that showed growth on a screen plate were inoculated on BHIA plates with rising vancomycin concentrations (1-20 mg/L). After subcultivation and growth on a vancomycin-free BHIA plate, minimal inhibitory concentrations (MICs) were determined for all strains. RESULTS: Fourteen of 48 (29.1%) strains had vancomycin 8 mg/L and 1/48 (2.0%) strain had vancomycin 16 mg/L. In 3/48 (6.2%) MIC were stable after storage in liquid nitrogen for six months. Vancomycin MIC50 and MIC90 of all 33 strains grown on screen plate were 1 and 2 gm/L, respectively, when tested on Mueller-Hinton agar (MHA) before inoculation on BHIA with growing concentrations of vancomycin. Immediately thereafter, MIC were 4 and 8 mg/L, and after six months of storage, they were 4 and 4 mg/L, respectively. CONCLUSION: The prevalence of hVISA in Croatia is low, but there are some strains with reduced susceptibility to vancomycin. Unfortunately, because of lack of clinical data neither clinical correlation with laboratory findings nor therapeutic failures can be discussed.