Prevalence, serogroup distribution and risk factors of Neisseria meningitidis carriage in high school and university students in Hungary.

Neisseria meningitidis causes life-threatening invasive meningococcal disease (IMD) with high mortality worldwide. Asymptomatic pharyngeal meningococcus colonisation is an important reservoir for the spread of the bacterium. The aim of this study was to determine N. meningitidis colonisation rates in asymptomatic high school and university students and to identify risk factors for carriage. Oropharyngeal swab samples and data from a self-reported questionnaire were obtained from overall 610 students, among them 303 university students and 307 high school students, aged between 15 and 31 years in Budapest, Hungary, between November 2017 and December 2018. Meningococcal carriage and serogroup of N. meningitidis were determined by RT-PCR from DNA extracted directly from the specimen. N. meningitidis was identified in 212 (34.8 %) of the participants. Significantly higher carriage rate was found among high school students (48.9 %) compared to university students (20.5 %). Peak of colonisation rate was among 17-19-year-old students (48.7 %). Most carriage isolates were non-typable (87.3 %). From the 212 meningococcus carriers, 19 were colonised by serogroup B (9 %), 5 by serogroup C (2.4 %), and 1 had serogroup Y (0.5 %). Significantly higher colonisation rate was found among males (42.4 %) than in females (33.1 %). Antibiotic use in the past 2 months has decreased the rate of meningococcal colonisation. Recent respiratory infection, active or passive smoking and attending parties have not influenced meningococcal colonisation rate significantly. In conclusion, we have found high asymptomatic meningococcus carriage rate among high school students and young adults, however, the majority of the colonizing meningococci were non-typable.

Coagulase-negative staphylococcal endophthalmitis: clinical severity and outcomes based on speciation.

OBJECTIVE: To identify characteristics and visual outcomes of coagulase-negative staphylococcal (CoNS) endophthalmitis in the era after the Endophthalmitis Vitrectomy Study. DESIGN: Single-centre retrospective analysis. PARTICIPANTS: Forty-two samples from 40 patients with documented CoNS endophthalmitis. METHODS: Visual acuity outcomes of CoNS endophthalmitis were assessed in relation to species and type of treatment instituted (i.e., pars plana vitrectomy [PPV] versus vitreous tap and injection of intravitreal antibiotics [T&I]) on 42 samples from 40 patients. RESULTS: Staphylococcus epidermidis was the most prevalent CoNS in our study. Cataract surgery and intravitreal injections were the most common sources for acute CoNS endophthalmitis. Eyes presenting with hand motion or better vision had similar mean final vision after either intravitreal antibiotics or PPV, whereas those with light perception or worse vision at onset had better outcomes after PPV only. Subanalysis showed that patients with S. epidermidis endophthalmitis (n = 39 eyes) had similar visual outcomes with either intravitreal injections or PPV regardless of visual acuity. Hypopyon and vitritis are not always present. CONCLUSIONS: Patients with S. epidermidis endophthalmitis may benefit similarly from either early vitrectomy or intravitreal antibiotic injections regardless of visual acuity. This finding may be a supplement to the complements the management standards set forth by the Endophthalmitis Vitrectomy Study.

Molecular microbiological testing of lower respiratory tract samples during COVID-19 pandemic / Alsó légúti minták molekuláris mikrobiológiai vizsgálata a koronavírus-járvány idoszakában

Introduction: BioFire FilmArray Pneumonia plus Panel (bioMerieux) is a PCR method for microbiological diagnostics of lower respiratory infections. It can detect 18 bacteria, 9 viruses and 7 antibiotic resistance genes in real time. It can help the differential diagnosis and the choice of therapy of pneumonia, by giving results in two hours. Objective: Reviewing the results of pneumonia PCR tests performed in our laboratory, and comparing them with the results of conventional culturing. Method: From October 2020 to September 2021, 820 lower respiratory tract samples were analyzed from inpatients with suspected pneumonia. Beside the PCR test, culturing was also performed. Oropharyngeal swabs were used for supplementary SARS-CoV-2 PCR. Results: 40% of samples were collected from SARS-CoV-2-positive patients. In 60% of the samples, the PCR test detected pathogens or resistance genes. The most commonly detected pathogens were Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii. 44% of the bacteria detected by PCR were not verified by culturing, whereas by culturing, several other bacteria, fungi and antibiotic resistance mechanisms were detected, which were not shown in the results of the multiplex PCR tests. In SARS-CoV-2-positive inpatients, 25.8% of the detected bacteria was S. aureus. The most common resistance gene was mecA/C (MRSA). In this group, other respiratory virus genes were detected in 2% of SARS-CoV-2-positive patients, whereas in 13% in samples of SARS-CoV-2-negative patients. Conclusions: Because of the importance of pathogens excluded from the PCR targets and multifactorial mechanisms of antibiotic resistance, culturing is recommended to perform beside pneumonia-specific multiplex PCR tests.

Similar Strains of Coagulase-Negative Staphylococci Found in the Gastrointestinal Tract and Bloodstream of Bacteremic Neonates.

OBJECTIVES: Premature neonates are susceptible to opportunistic and nosocomial infections. Efforts have been made to determine whether the neonatal gut microbiome possesses potential for causing bloodstream infections in newborns via microbial translocation from the gastrointestinal tract. We aimed to examine similarities in coagulase-negative staphylococci (CoNS) strains found in the gastrointestinal tract and bloodstream in bacteremic neonates. METHODS: CoNS strains isolated from blood cultures and perianal and pharyngeal swab samples of neonates from two neonatal intensive care units were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and pulsed-field gel electrophoresis. Molecular mass and genetic similarities of CoNS strains were compared. RESULTS: Marked similarity was found in the molecular mass and genetic profile of examined CoNS isolates from blood cultures and perianal/pharyngeal samples. The percentage of neonates developing bacteremia following perianal and pharyngeal colonization by CoNS was significantly higher when compared to those colonized by Enterobacteriales species (p < 0.0002). CONCLUSIONS: CoNS colonizing the gut may be a source of bacteremia in neonates. Enterobacteriales species do not contribute as significantly to bacteremia when compared to CoNS, and may be protective against gut mucosa-originated systemic infection.

Characterisation of antibiotic resistance, virulence, clonality and mortality in MRSA and MSSA bloodstream infections at a tertiary-level hospital in Hungary: a 6-year retrospective study.

BACKGROUND: Staphylococcus aureus bloodstream infections (BSI) cause significant morbidity and mortality due to the frequent antibiotic resistance, toxin and adhesin production of the bacterium. These characteristics differ significantly in methicillin resistant (MRSA) and methicillin sensitive S. aureus (MSSA) and also among isolates of different MRSA clones, contributing to the outcome of S. aureus bacteraemia. METHODS: In this study, all MRSA BSI isolates from Semmelweis University, Budapest, Hungary, isolated between 2011-2016 and the same number of matched MSSA (overall 306 isolates) were characterised in terms of antibiotic susceptibility, virulence genes, clonality and their association with all-cause 30-day mortality. Effect of patient related variables, such as age, gender and comorbidities were also investigated. RESULTS: ST22-MRSA-IV and ST5-MRSA-II were the most prevalent clones in our study. SCCmec I isolates showed the highest resistance rates and SCCmec II carried most virulence genes. Infections caused by SCCmec IV isolates were associated with the highest mortality rate (42.2%), despite the similar comorbidity rates of the different patient groups. All-cause 30-day mortality was 39.9% in the MRSA and 30.7% in the MSSA group. Increased teicoplanin MIC was associated with high mortality rate. Resistance to ciprofloxacin, erythromycin and clindamycin was common in MRSA, whereas MSSA isolates were more sensitive to all antibiotics with the exception of doxycycline. All MRSA isolates were sensitive to glycopeptides and linezolid; resistance to rifampicin and sulfamethoxazole-trimethoprim was low. MRSA isolates carried more adhesion genes, superantigens were more frequent in MSSA. Panton-Valentine leukocidin was found in 2.3% of the isolates. CONCLUSION: This study provides insight into the clonal composition and associated mortality of BSI S. aureus isolates in Hungary. The results suggest that the outcome of the infection is determined by the antibiotic resistance, genotype of the bacterium, and patient-related factors; rather than the virulence factors carried by the bacteria.

Ceftazidime-avibactam and ceftolozane-tazobactam susceptibility of multidrug resistant Pseudomonas aeruginosa strains in Hungary.

Our objective was to compare the activity ceftazidime-avibactam (C/A) and ceftolozane-tazobactam (C/T) against multidrug (including carbapenem) resistant Pseudomonas aeruginosa clinical isolates collected from six diagnostic centers in Hungary and to reveal the genetic background of their carbapenem resistance. Two hundred and fifty consecutive, non-duplicate, carbapenem-resistant multidrug resistant (MDR) P. aeruginosa isolates were collected in 2017. Minimal inhibitory concentration values of ceftazidime, cefepime, piperacillin/tazobactam, C/A and C/T were determined by broth microdilution method and gradient diffusion test. Carbapenem inactivation method (CIM) test was performed on all isolates. Carbapenemase-encoding blaVIM, blaIMP, blaKPC, blaOXA-48-like and blaNDM genes were identified by multiplex PCR. Of the isolates tested, 33.6% and 32.4% showed resistance to C/A and C/T, respectively. According to the CIM test results, 26% of the isolates were classified as carbapenemase producers. The susceptibility of P. aeruginosa isolates to C/A and C/T without carbapenemase production was 89% and 91%, respectively. Of the CIM-positive isolates, 80% were positive for blaVIM and 11% for blaNDM. The prevalence of Verona integron-encoded metallo-beta-lactamase (VIM)-type carbapenemase was 20.8%. NDM was present in 2.8% of the isolates. Although the rate of carbapenemase-producing P. aeruginosa strains is high, a negative CIM result indicates that either C/A or C/T could be effective even if carbapenem resistance has been observed.

A multicentre survey of the antibiotic susceptibility of clinical Bacteroides species from Hungary.

BACKGROUND: The species of the Bacteroides fragilis group are important components of human microbiota, but as opportunistic pathogens they can be the causative agents of severe infections. METHODS: The major aims of our investigation were the evaluation of the susceptibility of 400 different Hungarian B. fragilis group isolates to 10 antibiotics by the agar dilution method, the comparison of our resistance data with previous national and international antibiotic resistance data and the comparison of present data in regional aspect. The MIC-values on 10 antibiotics of all the strains were determined with the agar dilution method by CLSI. The presence of the cfiA gene in Division II B. fragilis strains was confirmed by RT-PCR. RESULTS: We detected a relatively high resistance rate of ampicillin, moxifloxacin, clindamycin and tetracycline, but amoxicillin/clavulanic acid, metronidazole, tigecycline and chloramphenicol showed excellent activity. In this study, we found that 6.75% of the isolates were resistant to cefoxitin and 7% to meropenem, while 8.58% of our B. fragilis strains harboured the cfiA gene. Most of the meropenem resistant strains were isolated in one of the participating centres. In the case of meropenem, cefoxitin, clindamycin and high-level-ampicillin-resistant strains, we found significant regional differences. DISCUSSION: Most of the results of our study were concordant with previous national and international data, with the exception of amoxicillin/clavulanic acid, cefoxitin and meropenem. CONCLUSIONS: Our study highlighted the importance of the periodic monitoring of the antimicrobial susceptibility of Bacteroides species providing important information for the appropriate therapy.

[Uncommon non-fermenting Gram-negative rods as pathogens of lower respiratory tract infection]. / Ritkábban eloforduló, alsó légúti fertozést okozó Gram-negatív nem fermentáló pálcák.

INTRODUCTION: Glucose non-fermenting Gram-negative bacteria are ubiquitous environmental organisms. Most of them are identified as opportunistic, nosocomial pathogens in patients. Uncommon species are identified accurately, mainly due to the introduction of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology practice. Most of these uncommon non-fermenting rods are isolated from lower respiratory tract samples. Their significance in lower respiratory tract infections, such as rules of their testing are not clarified yet. AIM: The aim of this study was to review the clinical microbiological features of these bacteria, especially their roles in lower respiratory tract infections and antibiotic treatment options. METHOD: Lower respiratory tract samples of 3589 patients collected in a four-year period (2013-2016) were analyzed retrospectively at Semmelweis University (Budapest, Hungary). Identification of bacteria was performed by MALDI-TOF MS, the antibiotic susceptibility was tested by disk diffusion method. RESULTS: Stenotrophomonas maltophilia was revealed to be the second, whereas Acinetobacter baumannii the third most common non-fermenting rod in lower respiratory tract samples, behind the most common Pseudomonas aeruginosa. The total number of uncommon non-fermenting Gram-negative isolates was 742. Twenty-three percent of isolates were Achromobacter xylosoxidans. Beside Chryseobacterium, Rhizobium, Delftia, Elizabethkingia, Ralstonia and Ochrobactrum species, and few other uncommon species were identified among our isolates. The accurate identification of this species is obligatory, while most of them show intrinsic resistance to aminoglycosides. Resistance to ceftazidime, cefepime, piperacillin-tazobactam and carbapenems was frequently observed also. CONCLUSIONS: Ciprofloxacin, levofloxacin and trimethoprim-sulfamethoxazole were found to be the most effective antibiotic agents. Orv Hetil. 2018; 159(1): 23-30.

Molecular characterisation of multidrug-resistant Bacteroides isolates from Hungarian clinical samples.

OBJECTIVES: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiota, however these bacteria can also cause severe infections. Due to frequent use of antibiotics, the spread of multidrug-resistant (MDR) strains is a real threat worldwide. METHODS: In a multicentre study, 400 Bacteroides isolates from five Hungarian microbiology laboratories were cultured and were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) of ten antibiotics were determined by the agar dilution method and were evaluated according to EUCAST or CLSI breakpoints. RESULTS: Six MDR strains were found and their antibiotic resistance genes were investigated by molecular methods The DNA amplicon of B. fragilis SZ38 was sequenced to search for a mutation in the gyrA gene. Among the six MDR isolates, one cfiA-, two cepA-, three cfxA-, two ermG-, six tetQ-, three tetX- and two bexA-positive strains were found. None of the MDR isolates harboured cepA, nim, ermB or tetX1 genes. CONCLUSIONS: In the past 12 years, only a few cases of MDR Bacteroides infections have been reported. Within a comprehensive multicentre survey, we demonstrated the relatively high prevalence of MDR strains isolated in one centre with five isolates as well as one isolate from another centre during a relatively short period of time. This study highlights the importance of antimicrobial susceptibility testing and surveillance among B. fragilis group isolates.

Colistin resistance among blood culture isolates at a tertiary care centre in Hungary.

OBJECTIVES: The emergence of colistin resistance has been detected worldwide in recent years. Whilst colistin susceptibility has been tested in carbapenem resistant Enterobacteriaceae as well as multidrug-resistant Pseudomonas spp. and Acinetobacter spp. during routine laboratory practice, the overall rate of colistin resistance was unknown in our centre. The aim of this retrospective study was to reveal the prevalence of colistin resistance among clinically significant blood culture isolates in two different periods (2010-2011 and 2016) in our laboratory. METHODS: Consecutive non-duplicate strains (n=776) were screened for colistin resistance using agar plates containing 4mg/L colistin. Strains cultured on colistin-containing plates were further examined. Minimum inhibitory concentrations (MICs) of colistin-tolerant subcultures and original cultures were determined in parallel by the broth microdilution method. Screening for mcr-1-mediated colistin resistance was performed by PCR. RESULTS: The rate of colistin resistance was 0.6%, 1.3% and 2.6% in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., respectively; colistin-resistant subpopulations were found in 17%, 27% and 20% of isolates, respectively, with low frequency. Seven colistin-resistant strains were found, among which was an mcr-1-positive Escherichia coli isolated from a blood sample of a haemato-oncology patient in 2011. All Stenotrophomonas maltophilia isolates were resistant to colistin. CONCLUSIONS: The low prevalence of colistin resistance was in accordance with European data. The prevalence of heteroresistance was significantly higher, but the clinical significance of the phenomenon is unclear. We have identified the first mcr-1-positive E. coli strain in Hungary. mcr-1 has been in Hungary since 2011 but has not yet expanded.

Does Staphylococcus Saprophyticus Cause Acute Cystitis only in Young Females, or is there more to the Story? A One-Year Comprehensive Study Done in Budapest, Hungary.

Staphylococcus saprophyticus is a well-known urinary pathogen in acute cystitis in young females. We completed a retrospective overview of the distribution of urinary tract infections (UTIs) occurring in 2014, at Semmelweis University hospitals and at Heim Pál Children's Hospital. Six age-groups (ages 0-100) were examined, with the frequency of S. saprophyticus in females being: 0.1% (0-4), 0.7%, (5-15), 7.4% (16-24), 1.2% (25-39), 0.4% (40-59) and 0.1% (60-100), and S. saprophyticus being the 3(rd) most common pathogen in females aged 16-24. In males, S. saprophyticus was only isolated from those aged 5-15. Seasonal distribution of UTIs caused by S. saprophyticus showed that most infections occurred during the months of January, June, August and November. Antibiotic-resistance rates of amoxicillin, clindamycin, doxycycline, erythromycin, gentamicin and sulfamethoxazole- trimethoprim varied as follows: 0.9%, 32.7%, 19.6%, 34.6%, 0.9% and 0.9%, respectively. Thirty randomly selected samples were analysed by pulsed-field gelelectrophoresis, and 28 different genotypes were identified. S. saprophyticus is involved in the pathogenesis of acute cystitis not only in young females, but also in other age-groups, and in young males as well. We did not find any significant seasonal occurrence in S. saprophyticus-caused UTIs. The infective strains were genetically diverse. Antibiotic-resistance does not pose any issue as of yet.

In vitro biofilm production of Candida bloodstream isolates: any association with clinical characteristics?

Candida spp. are a leading cause of bloodstream infection (BSI) and are associated with high mortality rates. Biofilm production is a virulence factor of Candida spp., and has been linked with poor clinical outcome. The aim of our study was to assess biofilm production of Candida bloodstream isolates at our institute, and to determine whether in vitro biofilm production is associated with any clinical characteristics of infection. During the four-year study period, 93 cases of Candida BSI were analysed. The most frequently isolated species was C. albicans (66.7 %), followed by C. glabrata (9.7 %), C. parapsilosis (9.7 %), C. tropicalis (9.7 %) and C. krusei (4.3 %). Biofilm production was more prevalent among non-albicans Candida spp. (77.4 %) than C. albicans (30.6 %) (P = 0.02). Abdominal surgery was identified as a risk factor of BSI caused by biofilm producing non-albicans Candida isolates. No risk factors predisposing to bloodstream infection caused by a biofilm producing C. albicans isolate were identified. Biofilm production was not verified as a risk factor of mortality.

Antibiotic susceptibility of sulfamethoxazole-trimethoprim resistant Stenotrophomonas maltophilia strains isolated at a tertiary care centre in Hungary.

Sulfamethoxazole-trimethoprim (SXT) is the drug-of-choice in Stenotrophomonas maltophilia caused infections. There has been an increase in resistance to SXT of S. maltophilia over recent years. In this study 30 S. maltophilia clinical isolates resistant to SXT were investigated. Antibiotic susceptibilities for ciprofloxacin, moxifloxacin, levofloxacin, doxycycline, tigecycline, ceftazidime, colistin and chloramphenicol were determined by broth microdilution method. None of the strains were susceptible to ciprofloxacin, tigecycline, ceftazidime or colistin. Only 37% of the isolates were susceptible to levofloxacin or moxifloxacin. Two isolates resistant to all tested antibiotic agents and two others susceptible only to doxycycline were further investigated: susceptibility for combinations of antibiotics was analyzed by checkerboard technique. According to the fractional inhibitory concentration indices calculated, moxifloxacin plus ceftazidime combination was found to be synergistic in each case. Genetic testing revealed the predominance of sul1 gene. Our study concluded that the range of effective antibiotic agents is even more limited in infections caused by SXT-resistant S. maltophilia. In these cases, in vitro synergistic antibiotic combinations could be potential therapeutic options.

Significance of yeasts in bloodstream infection: Epidemiology and predisposing factors of Candidaemia in adult patients at a university hospital (2010-2014).

The incidence of Candida bloodstream infection (BSI) has increased during the past decades. Species distribution is changing worldwide, and non-albicans Candida spp. are becoming more prevalent. Acquired resistance to antifungal agents has been documented in several reports. The aim of our study was to assess the epidemiology and antifungal susceptibility of Candida isolates from BSI at our institute. The incidence of Candida BSI increased during the first four years of our investigation, from 1.7 to 3.5 episodes / 10 000 admissions, then dropped to 2.66 episodes / 10 000 admissions in the last year. The most frequently isolated species was C. albicans (63%), followed by C. glabrata (13%), C. parapsilosis (10.2%), C. tropicalis (9.3%), and C. krusei (3.7%). One isolate each of C. kefyr, C. fabianii and C. inconspicua were detected. The percentage of C. albicans remained stable throughout the study period. The most frequent risk factors of Candida BSI in our patient population were intensive care treatment (60.4%), abdominal surgery (52.5%), and solid malignancy (30.7%). All isolates were wild-type organisms, no acquired antifungal resistance was detected.

[Special application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiological diagnostics]. / Mátrix-asszisztált lézer deszorpciós, ionizációs, repülési ido mérésén alapuló tömegspektrometria speciális alkalmazása a klinikai mikrobiológiai diagnosztika területén.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a new possibility for rapid identification of bacteria and fungi revolutionized the clinical microbiological diagnostics. It has an extreme importance in the routine microbiological laboratories, as identification of the pathogenic species rapidly will influence antibiotic selection before the final determination of antibiotic resistance of the isolate. The classical methods for identification of bacteria or fungi, based on biochemical tests, are influenced by many environmental factors. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a rapid method which is able to identify a great variety of the isolated bacteria and fungi based on the composition of conserved ribosomal proteins. Recently several other applications of the method have also been investigated such as direct identification of pathogens from the positive blood cultures. There are possibilities to identify bacteria from the urine samples in urinary tract infection or from other sterile body fluids. Using selective enrichment broth Salmonella sp from the stool samples can be identified more rapidly, too. The extended spectrum beta-lactamase or carbapenemase production of the isolated bacteria can be also detected by this method helping the antibiotic selection in some cases. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry based methods are suitable to investigate changes in deoxyribonucleic acid or ribonucleic acid, to carry out rapid antibiotic resistance determination or other proteomic analysis. The aim of this paper is to give an overview about present possibilities of using this technique in the clinical microbiological routine procedures.

Infection and colonization by Stenotrophomonas maltophilia: antimicrobial susceptibility and clinical background of strains isolated at a tertiary care centre in Hungary.

BACKGROUND: Stenotrophomonas maltophilia is an important opportunistic, mainly nosocomial pathogen that emerged in the last decades worldwide. Due to its inherent extended antibiotic resistance, therapeutic options are strongly limited. New resistance mechanisms in S. maltophilia make antibiotic therapy even more difficult. The aim of our study was to investigate the antimicrobial resistance of S. maltophilia isolates collected in our laboratory and to reveal related clinical background. METHOD: Consecutive non-duplicate S. maltophilia isolates (n = 160) were collected in a three-year period. Conventional methods, automated identification system and MALDI-TOF MS was used for identification, ERIC-PCR for genetic relationship analysis and broth microdilution method to determine the susceptibility for trimethoprim/sulfamethoxazole (SXT), ciprofloxacin, levofloxacin, moxifloxacin, colistin, doxycycline and tigecycline. Clinical final reports were used retrospectively to collect clinical information. RESULTS: ERIC-PCR revealed large heterogeneity. Trimethoprim/sulfamethoxazole, moxifloxacin and levofloxacin were found to be the most effective agents with MIC50/MIC90 0.5/1, 0.25/1, 1/2 mg/l, respectively. Seventy percent of patients with S. maltophilia infection were treated in intensive care units. All-cause mortality rate was 45%. Nearly 70% of the isolates were collected from polymicrobial infections/colonizations. CONCLUSIONS: Trimethoprim/sulfamethoxazole is the most potent antibiotic agent against S. maltophilia. In case of SXT hypersensitivity, intolerance or resistance, fluoroquinolones are alternative therapeutic options. Missing clinical breakpoints, consensus antibiotic susceptibility testing guidelines and clinical trials make the interpretation of antibiotic susceptibility testing results difficult. The indirect pathogenicity of S. maltophilia in polymicrobial infections or colonizations has to be taken into consideration.

Emergence of VIM-4- and SHV-12-producing Enterobacter cloacae in a neonatal intensive care unit.

In order to reveal colonization with multidrug-resistant bacteria early, routine screening is done on samples of all patients of the neonatal intensive care units at Semmelweis University, Hungary. Due to the extended-spectrum ß-lactamase (ESBL) screening examinations, emergence of multidrug-resistant Enterobacter cloacae isolates was found with suspicion of clonal transmission, therefore active microbiological surveillance was initiated. The aim of our study was to characterize 60 E. cloacae isolates recovered in a 7-month period in 2010. MIC values of antibiotics were determined and ESBL and carbapenemase production was tested. Metallo-ß-lactamase (MBL) genes, ESBL genes, and class-1 integrons were characterized, and the possible clonal relationship between isolates was investigated. The isolates showed increased MIC values for carbapenems and cephalosporins. All 60 E. cloacae strains recovered from 16 neonates proved to be VIM-4 MBL producers. Fifty-three strains were SHV-12 ESBL producers also. In all cases, the bla(VIM-4) gene was a part of class-1 integron, In238a. XbaI-macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) revealed identical patterns for the isolates. Our study supports the importance of active microbiological surveillance as well as molecular epidemiology at the NICUs as a part of infection control.

The Consequence of a Founder Effect: CCR5-∆32, CCR2-64I and SDF1-3'A Polymorphism in Vlach Gypsy Population in Hungary.

Frequencies of genetic polymorphisms of the three most frequent HIV-1 resistance-conferring alleles playing an important role in HIV-1 pathogenesis were analysed in Vlach Gypsy populations living in Hungary, as the largest minority. Mutations in the encoding genes, such as CCR5-∆32, CCR2-64I and SDF1-3'A are shown to result in protective effects against HIV-1 infection and disease progression. 560 samples collected from Vlach Gypsy individuals living in 6 North-East Hungarian settlements were genotyped by PCR-RFLP method. Overall allele frequencies of CCR5-∆32, CCR2-64I and SDF1-3'A were found as 0.122, 0.186 and 0.115 respectively. All the observed genotype frequencies were in accordance with Hardy-Weinberg equilibrium . In regions, however, Vlach Gypsies live in majority and in ethnically homogenous communities, a higher CCR5-∆32 mutations were found, with allele frequencies of 0.148 and 0.140 respectively, which are remarkably higher than those in general Hungarian people, and ten times higher than in regions of North-Western India from where present day Hungarian Gypsies originated in the Middle Ages. In the background of this higher CCR5-∆32 allele frequency in the population analysed in our study a genetic founder effect could be assumed. Allele frequency of CCR2-64I was found to be among the highest in Europe. SDF1-3'A allele frequency in Vlach Gypsies was significantly lower than in ethnic Hungarians. 63% of the total 560 individuals tested carried at least one of the mutations studied. These results could partially explain the low incidence of HIV/AIDS among Vlach Gypsies in Hungary.

HVS-I polymorphism screening of ancient human mitochondrial DNA provides evidence for N9a discontinuity and East Asian haplogroups in the Neolithic Hungary.

Analysis of mitochondrial mutations in the HVS-I region is an effective method for ancient human populational studies. Discontinuous haplotype data between the first farmers and contemporary Europeans has been described before. Our contribution is based on a survey initiated on the Neolithic skeletons from Hungarian archaeological sites in the Alföld. This Lowland, the Hungarian Plain, is well excavated as an important region for spread of Neolithic culture from Near East and Balkans toward Central and Western Europe, started circa 8000 years ago. HVS-I sequences from nt15977 to nt16430 of 11 such specimens with sufficient mitochondrial DNA preservation among an extended Neolithic collection were analysed for polymorphisms, identifying 23 different ones. After assigning all single-nucleotide polymorphisms, a novel, N9a, N1a, C5, D1/G1a, M/R24 haplogroups were determined. On mitochondrial control mutations at nt16257 and nt16261, polymorphic PCRs were carried out to assess their distribution in remains. Neolithic data set was compared with contemporary Vác samples and references, resulting in higher frequency of N9a in Alföld as a remarkable genetic discontinuity. Our investigation is the first to study mutations form Neolithic of Hungary, resulting in an outcome of Far Eastern haplogroups in the Carpathian Basin. It is worth further investigation as a non-descendant theory, instead of a continuous population history, supporting genetic gaps between ancient and recent human populations.

Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers.

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.