AB Biodisk Research Articles

Surveillance of ESBL-producers causing UTIs over a 6 year period

Background: Urinary tract infections (UTIs) are one of the most prevalent community-acquired infections. ESBL-producing microorganisms are a growing threat to public health, and their presence among the community is spreading. The aim of this study was to access the prevalence of ESBL-producing isolates collected from urine of elderly attending the Emergency Room (ER) during a 6 year period (2011-2016). Methods & Materials: Bacterial strains were isolated from patients with primary diagnose UTI, older than 65 years, in the ER between 2011 and 2016. The Vitek2 system (according to EUCAST 2014 guidelines) and Advanced Expert System (BioM~rieux, Marcy L'toile, France) were used to identify and perform the antibiotic susceptibility profile of the isolates. ESBL production was confirmed using the Etest(r) (AB Biodisk) method, according to the manufacturer instructions. Results: 2925 samples were collected following the criteria listed above. The percentage of Escherichia coli isolates has been increasing since 2011, up to 51,7% in 2016, and the percentage of Klebsiella pneumoniae, which in 2011 was higher than E. coli, has been significantly decreasing in the last years, to around 15%. A reduction in the percentage of ESBL-producers until 2013 was noted, from where there has been an increase, both among E. coli and K. pneumoniae. In 2011, 82,5% of K. pneumoniae were ESBL-producers, and progressively decreases until 2015 to 32,5%. But a slightly increase in 2014 and 2016 (44,1% and 51,2%) shows the tendency of ESBL-producing K. pneumoniae spread in the community. E. coli ESBL-producers have also progressively diminished until 2013, then exhibiting an increase up to 24,0% in 2016. Conclusion: Considering the criteria used, we can conclude that they are carried by the patients who attend the ER repeatedly, thus spread in the community. Despite ESBL-producers have been significantly reduced in the last years, results show a possible increasing tendency. The same is observed both for E. coli and K. pneumoniae isolates, where the decrease in ESBL-producers until 2013 is now being overcome by higher percentages. Nevertheless, 2016 numbers are still high, being a motive of concern especially considering the elderly population.

Defining of gene mutations and minimal inhibitory concentrations in isoniazid resistant Mycobacterium tuberculosis isolates

Tuberculosis is a very important disease all over the world despite the advanced diagnostic and treatment regimens. Resistant tuberculosis, which has increased in recent years in particular, is a global problem and prevents the fight against tuberculosis. For this reason, it is important to determine the etiologic agent early and its sensitivity against antituberculosis drugs. Resistance profiles of the isolates and the gene mutations causing resistance are determined for epidemiological purposes and mutation regions of the isolates are being investigated on the basis of countries. The aim of our study was to determine the minimal inhibitor concentration (MIC) of isoniazid (INH) and to investigate the relationship between mutations in resistance genes and MIC values. For this purpose, 25 isoniazid (INH) monoresistant and 25 multidrug resistant (MDR), in total 50 clinical isolates were used and gene mutations causing INH resistance and the relationship of these mutations with minimal inhibitor concentrations (MICs) were searched by GenoType MTBDRplus (Hain Lifescience GMBH, Nehren, Germany) and antibiotic gradient test (E-test, AB BIODISK, Solna, Sweden) methods. The concordance of GenoType MTBDRplus and antibiotic gradient test methods for INH sensitivity, was 88% in INH monoresistant isolates, 80% in MDR isolates and 84% in all isolates. The most frequent mutation zone in INH monoresistant isolates was inhA C15T promotor zone (12 isolates, 54.5%) however, MIC of INH was > 256 µg/ml in two isolates and mutation in katG S315T was observed in both of these isolates. The most frequent mutation zone in MDR isolates was katG S315T (14 isolates, 56%) codon. MIC of INH was > 256 µg/ml in 10 isolates and mutation was observed in katG S315T codon in 5 (50%) isolates, in katG S315T codon and inhA C15T promotor zone in 3 (30%) isolates and in inhA promotor zone in 2 (20%) isolates, respectively. When all the isolates were analyzed, the most frequent mutation was found in katG S315T codon (24 isolates, 48%), MIC of INH was > 256 µg/ml among 12 isolates and the most frequent mutation zone in these isolates which have high MIC for INH was katG S315T codon. In conclusion, the results of this study have shown that the value of MICs for INH is high in isolates with katG S315T mutation, nevertheless, further investigations are needed to support this result.

Evaluation of different phenotypic tests for detection of metallo-β-lactamases in imipenem-resistant Pseudomonas aeruginosa.

PURPOSE:Pseudomonas aeruginosa causes a wide spectrum of infections including bacteremia, pneumonia, urinary tract infection, etc., Metallo-beta-lactamase (MBL) producing P. aeruginosa is an emerging threat and cause of concern as they have emerged as one of the most feared resistance mechanisms. This study was designed to know the prevalence of MBL production in P. aeruginosa and to evaluate the four phenotypic tests for detection of MBL production in imipenem-resistant clinical isolates of P. aeruginosa.METHODS:Totally, 800 isolates of P. aeruginosa isolated from various clinical samples were evaluated for carbapenem resistance and MBL production. All imipenem-resistant strains were tested for carabapenemase production by modified Hodge test. Screening for MBL production was done by double-disc synergy test and combined disc test (CDT). Confirmation of MBL production was done by the E-test (Ab BioDisk, Solna, Sweden).RESULTS:Out of the 800 isolates of P. aeruginosa, 250 isolates were found resistant to imipenem. Based on the results of E-test, 147 (18.37%) isolates of P. aeruginosa were positive for MBL production. The CDT has the highest sensitivity and specificity for the detection of MBL production as compared to other tests.CONCLUSION:The results of this study are indicative that MBL production is an important mechanism of carbapenem resistance among P. aeruginosa. Use of simple screening test like CDT will be crucial step toward large-scale monitoring of these emerging resistant determinants. Phenotypic test for MBL production has to be standardized, and all the isolates should be routinely screened for MBL production.

Antimicrobial susceptibility of Clostridium difficile isolated from food animals on farms

Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. C. difficile can also inhabit healthy food animals and transmission to humans is possible. As a result of the complexity and cost of testing, C. difficile is rarely tested for antimicrobial susceptibility. A total of 376 C. difficile strains (94 each from swine and dairy feces, and 188 from beef cattle feces) were isolated from healthy food animals on farms during studies conducted by the National Animal Health Monitoring System. Using the Etest (AB Biodisk, Solna, Sweden), samples were tested for susceptibility to nine antimicrobials implicated as risk factors for CDAD (linezolid, amoxicillin-clavulanic acid, ampicillin, clindamycin, erythromycin, levofloxacin, metronidazole, rifampicin, and vancomycin). Vancomycin was active against all isolates of C. difficile (MIC90=3.0μg/ml) while almost all isolates (n=369; 98.1%) were resistant to levofloxacin. With the exception of vancomycin, resistance varied by animal species as follows: linezolid (8.5% resistance among swine versus 2.1 and 1.1% resistance among dairy and beef, respectively), clindamycin (56.4% resistance among swine versus 80% and 90.9% resistance among dairy and beef, respectively), and rifampicin (2.1% and 0% resistance among swine and dairy cattle isolates, respectively versus 14.3% resistance among beef isolates). Regardless of species, multiple drug resistance was observed most often to combinations of clindamycin and levofloxacin (n=195; 51.9%) and ampicillin, clindamycin and levofloxacin (n=41; 10.9%). The reason for the variability of resistance between animal species is unknown and requires further research.

Evaluation of antimicrobial resistance of Helicobacter pylori in the last 15 years in West Poland.

Increasing resistance to drugs represents a serious problem in treatment of infections with Helicobacter pylori, providing cause of frequent therapeutic failures. Present study aimed at analysis of changes in resistance of H. pylori to antibiotics in West Poland within the recent 15 years. 108 strains of H. pylori were analysed, isolated from gastric mucosa of adult patients. Group 1 involved 66 strains isolated in years of 1998/1999. Group 2 comprised 42 isolates obtained in years of 2013/2014. Susceptibility to amoxicillin (AMX), clarithromycin (CL), tetracycline (TC) and metronidazole (MTZ) was determined by E-test (AB Biodisc). All strains on both studied groups were susceptible to AMX. In group 1 all strains proved to be susceptible to TC, while 9% and 36% of tested strains were resistant to CL and MTZ, respectively. By contrast, in group 2, 31% and 83% of strains were resistant to CL and MTZ, respectively. In parallel, 14% strains were found to be resistant to TC (according to EUCAST interpretations). In West Poland, within recent 15 years a dramatic increase was noted in H. pylori strains resistant to metronidazole. In parallel, a significant increase was noted in proportion of strains resistant to clarithromycin.

Investigation of beta-lactamase genes and clonal relationship among the extended-spectrum beta-lactamase producing nosocomial Escherichia coli isolates

Extended-spectrum beta-lactamase (ESBL) producing microorganisms currently cause a major problem. Among theseCTX-M beta-lactamase producing Escherichia coli has also disseminated worldwide as an important cause of both nosocomial and community-acquired infections. The aims of this study were to determine the prevalence of the beta-lactamase genes, antibiotic susceptibilities and clonal relationships of ESBL-producing nosocomial E.coli isolates. A total of 76 ESBL-producing E.coli strains isolated from urine (n= 26), blood (n= 25) and wound (n= 25) specimens of hospitalized patients identified as nosocomial infection agents according to the CDC criteria between June 2010-June 2011 were included in the study. Antibiotic susceptibilities of the isolates were detected by Kirby-Bauer disc diffusion method according to CLSI recommendations. ESBL production was tested by double disc diffusion method, and cefotaxime/cefotaxime-clavulanic acid E-test strips (AB Biodisk, Sweden) were used for indeterminate results. Presence of TEM, SHV, CTX-M, OXA-2 group, 0XA-10 group, PER, VEB and GES beta-lactamase genes were investigated by polymerase chain reaction (PCR) using specific primers. Pulsed-field gel electrophoresis (PFGE) method was used for the detection of clonal relationships among the strains. Most of the ESBL-producing E.coli strains were isolated from samples of inpatients in intensive care (35%), internal medicine (16%) and general surgery (13%) units. All of the 76 strains were found susceptible to imipenem, meropenem and amikacin; however all were resistant to cefotaxime and ceftriaxone. The susceptibility rates of the isolates to cefoxitin, ertapenem, cefoperazone/sulbactam, piperacillin-tazobactam, gentamicin, ciprofloxacin, cefepime, amoxicillin-clavulanic acid, aztreonam and ceftazidime were 96%, 83%, 63%, 61%, 50%, 41%, 25%, 21%, 20% and 18%, respectively. Among E.coli isolates, the frequency of CTX-M, TEM, OXA-2 group, PER, SHV and OXA-10 group beta-lactamase genes were found as 89.5%, 59.2%, 15.8%, 14.5%, 11.8% and 3.9%, respectively, while none of the isolates were positive for VEB and GES beta-lactamase genes. In 1 (1.3%) strain none of the investigated genes were detected. PCR analyses of the isolates revealed that 25 harbored CTX-M and TEM genes together, while 20 harbored only CTX-M and two harbored only TEM genes. Single SHV gene was not detected in any of the isolates. PFGE demonstrated no major clonal relationship between ESBL-producing isolates. This study indicated that CTX-M type enzymes were highly endemic among ESBL-producing nosocomial E.coli strains in our hospital, with the polyclonal spread of ESBL-producing bacteria without any dominant epidemic clone. In conclusion, it was considered that further studies are needed to explain the relationship between epidemic clones and plasmids with the use of plasmid analysis and multilocus sequence typing methods.

Gastrointestinal Colonization with Vancomycin-Resistant Enterococci In Hospitalized and Outpatients.

BACKGROUND:The incidence of infection and intestinal colonization with vancomycin resistant enterococci (VRE) is increasing in many countries in the last decade. Concerning the difficult antimicrobial treatment of infections caused by VRE, decreasing the incidence and prevalence of these infections is an important factor in VRE-induced morbidity and mortality control.AIM:To determine the prevalence of gastrointestinal colonization with vancomycin resistant enterococci in hospitalized and outpatients, and to determine the genetic base of the vancomycin resistance in VRE isolates.MATERIAL AND METHODS:Seven hundred and eighty stool specimens were investigated for the gastrointestinal carriage of vancomycin-resistant enterococci (VRE). Susceptibility to vancomycin was tested in all isolates by disk-diffusion test and E-test (AB Biodisk, Sweden). Determined vancomycin resistant enterococci were than tested for detection of vanA, vanB and vanC genes by PCR.RESULTS:Vancomycin resistant strains of enterococci were isolated from 46 (16.1 %) of the 285 hospitalized patients and 5 (7.7 %) of the 65 patients living in the community (p < 0.05). The most of the highly resistant enterococci strains to vancomycin (95.2 %), were identified as E. faecium. Minimal inhibitory concentrations (MICs) to vancomycin in all 39 vanA genotypes of E. faecium and two vanA genotypes of E. fecalis were > 256 μg/ml. Three vanB genotypes of E. faecium and one vanB genotype of E. faecalis had MICs of 32 μg/ml. All six vanC genotypes of E. gallinarum had MICs of 8 μg/ml. All vanA genotypes of VRE were highly resistant to vancomycin, with MICs above 256 μg/ml. Three vanB genotypes of VR E. faecium and one VR E. fecalis were resistant, with MICs 32 μg/ml. vanC genotypes of VR E. gallinarum were intermediate resistant to vancomycin with MICs of 8 μg/ml.CONCLUSIONS:The prevalence of vancomycin resistant enterococci in Republic of Macedonia was 2-fold higher in hospitalized than in outpatients. VanA genotype was dominant in isolates of E. faecium and it was highly associated with the MIC values above the 256 μg/ml. Since most of the enterococcal infections are endogenous, there is a need for screening the colonization of patient’s intestinal flora with VRE at the hospital entry. Identification and genotyping of faecal enterococci, together with their susceptibility testing to vancomycin, could be useful marker for the infection control.

Successful containment of carbapenem-resistant Enterobacteriaceae by strict contact precautions without active surveillance

Carbapenem-resistant Enterobacteriaceae (CRE) are a growing problem worldwide. Guidelines focus on carbapenemase-producing organisms, and little is known about whether strict adherence to infection control measures is effective for CRE without carbapenemase. During 2009, CRE increased markedly in a tertiary hospital, and enhanced infection control measures without active surveillance were adopted. Beginning in April 2010, enhanced antimicrobial stewardship, strict contact precautions, and cohort isolation were adopted. After September 2010, hand hygiene performance was prospectively monitored by active surveillance, and results were monthly fed back to medical personnel. Available carbapenem-resistant Escherichia coli (ECO) and carbapenem-resistant Klebsiella pneumoniae (KPN) isolated during 2008-2010 were characterized. Imipenem and meropenem minimal inhibitory concentrations were confirmed by E-test (AB biodisk, Solna, Sweden). Phenotypic screening assays and polymerase chain reaction (PCR) amplification of known β-lactamase and carbapenemase genes were performed. From 3,511 ECO and 2,279 KPN, 44 (0.76%) were CRE (3 ECO, 41 KPN). CRE incidence rates rose from 1.61 in 2008 to 5.49 in 2009; they rose further to 9.81 per 100,000 patient days in early 2010. After adoption of strict infection control measures, CRE frequency fell back in 2011 and remained at baseline afterward. Phenotypic screening and PCR showed AmpC β-lactamase and extended spectrum β-lactamases with or without loss of porins; carbapenemases were not detected. Enhanced infection control measures, even without active surveillance, seem effective to prevent further spread of CRE in a low-prevalence setting with mainly carbapenemase-nonproducing CRE.

Multi-drug carbapenem-resistant Klebsiella pneumoniae infection carrying the OXA-48 gene and showing variations in outer membrane protein 36 causing an outbreak in a tertiary care hospital in Riyadh, Saudi Arabia

To investigate the genes of antibiotic resistance among isolates from the first reported carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak in a tertiary care hospital, Riyadh, Saudi Arabia. Antimicrobial susceptibility testing was performed on bacterial isolates using the Microscan Walkaway system (Siemens, Germany) and was confirmed by Etest (AB Biodisk, Sweden). bla-CTX-M, -SHV, -TEM, -OXA-48, OXA-A,B,C,D, -KPC, -NDM, -VIM, -IMP, integron 1, and outer membrane proteins(Omp)-35 and Omp-36 were investigated by PCR amplification and direct sequencing of PCR products. Isolates were sequence-typed by multilocus sequence typing (MLST). All isolates were resistant to cefotaxime, ceftazidime, cefepime, ciprofloxacin, and piperacillin-tazobactam, and 91% (21 out of 23) were resistant to amikacin and gentamicin. All isolates except two from a single patient were resistant to one of the carbapenems. CTX-M and SHV genes were detected in all isolates, CTX-M-15 and SHV-1 types being predominant among these extended-spectrum beta-lactamases (ESBLs). TEM-1 was found in all except one isolate (isolate 3). Significantly, the OXA-48 gene was also found in all isolates. OXA-D-gene was found in three out of 23 isolates. KPC, NDM, OXA-A, -B, -C, VIM, and IMP genes were absent in all isolates. Disruption of the Omp-36 gene due to insertion of transposon IS903 and/or IS4 was detected in four out of 23 isolates, and some unique variations were also observed in this gene, including an insertion of two amino acids in the L3 region of Omp-36 in one isolate (isolate 3) and a mutation resulting in a premature stop codon in another isolate (isolate 25). MLST revealed ST29 to be the predominant sequence type (17 out of 23 isolates, 74%). Three were ST709 and one each was ST37 and ST111; one isolate had an unknown ST. This is probably the first reported outbreak of multidrug/carbapenem-resistant Klebsiella infection involving the OXA-48 gene from Saudi Arabia. Although the presence of ESBLs such as OXA, CTX-M, TEM, and SHV are predictable reasons for resistance, variations in the Omp-36 gene might also have precipitated this phenomenon. Disruption of the Omp-36 sequence by large insertional elements, the insertion of two amino acids in a very crucial part of this protein, and the presence of a premature stop codon in one isolate might have rendered this protein incomplete and non-functional. The study also demonstrated that more than one type of clone was responsible for this reported apparent outbreak and that ST29, a clone not reported from this region before, was the major clone responsible.

Klinik Örneklerden İzole Edilen Acinetobacter baumannii Suşlarında Beta-Laktamaz Kaynaklı Direncin Moleküler Karakterizasyonu

Acinetobacter baumannii is an important cause of nosocomial infections that particularly increase the mortality and the morbidity at the intensive care units of the hospitals. The aims of this study were to evaluate the resistance genes, antibiotic susceptibility and the clonal relations among Acinetobacter strains isolated from clinical samples and to determine the resistance mechanisms related to these bacteria in our hospital. A total of 201 A.baumannii strains isolated from different clinical samples (35.3% from tracheal aspirate, 27.3% from blood, 18.4% from abscess material, 19% from other samples) of 160 inpatients evaluated at the Ibni Sina Hospital Central Bacteriology Laboratory, Ankara University School of Medicine, Turkey from April 2010 to December 2011, were included in the study. Identification of the isolates and their susceptibility testing against amikacin, ciprofloxacin, tetracycline, sulbactam/ampicillin, trimethoprim/sulfametoxazole (SXT), ceftazidime, gentamicin, imipenem, levofloxacin, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam, cefepime and colistin were performed by the automated systems, namely Vitek 2 (bioMérieux, France) and BD Phoenix (Becton Dickinson, USA). The molecular mechanisms of beta-lactamase resistance and the presence of integrons were analyzed by polymerase chain reaction (PCR). Moreover, since blaPER-1 gene is of high frequency in Turkey, it was also investigated in the isolates. Pulsed-field gel electrophoresis (PFGE) was performed to examine the clonal relations between isolates. Our results indicated that multidrug resistance rate of A.baumannii was 94.5% (190/201), while 94% (189/201) of the isolates were susceptible to colistin thus making it the most potent antimicrobial agent, followed by amikacin and SXT with a susceptibility rate of 32%. Twelve colistin-resistant isolates were further investigated with the E-test method (AB Biodisk, Sweden) and found to be colistin-resistant. While the results were negative for the genes responsible from metallo-beta-lactamase production, positive results were obtained for blaOXA genes at various rates (OXA-51 100%; OXA-23 91.5%; OXA-58 7%; OXA-24 2%). PFGE results revealed four different main clones (29 isolates in genotype A, 23 in genotype B, 18 in genotype C and 7 in genotype D) in the study population. No common epidemic isolate was detected. Class 1 integrons which take part in the transfer of resistance genes were detected in 112 (55.7%) isolates. There was no statistically significant difference between the genotype distributions of class 1 integron positive strains (p> 0.05). The relationship between the presence of integron in multidrug resistant isolates and resistance to tetracyclin, SXT, imipenem, meropenem, cefoperazone/sulbactam and cefepime were found to be statistically significant (p< 0.05). Of the isolates 42 (21%) were positive for blaPER- 1 gene and all were resistant to ceftazidime. This study indicated that blaOXA genes found together with blaOXA-51 genes play an important role in carbapenem resistance of A.baumannii strains. Moreover, multidrug resistance is still an important problem in infections caused by A.baumannii and integrons play a role in the transfer of the resistance genes. In conclusion, multidrug resistant A.baumannii strains were common in our hospital and our epidemiologic data would be helpful for further investigations and in therapeutical approaches.

Investigation of In Vitro Activity of Five Antifungal Drugs against Dermatophytes Species Isolated from Clinical Samples Using the E-Test Method.

Dermatomycosis is an infection with fungi related to the skin: glabrous skin, hair and/or nails. Oral treatment of fungal infections in dermatology has become a preferred modality for the management of these very common conditions. Although there are increasing numbers of antifungals available for treatment of dermatophytes, some cases and relapses have been unresponsive to treatment. The determination of fungus in-vitro antifungal susceptibility has been reported to be important for the ability to eradicate dermatophytes. It is necessary to perform antifungal susceptibility testing of dermatophytes. E-test (AB Biodisk, Sweden) is a rapid, easy-to-perform in-vitro antifungal susceptibility test. The aim of this study was to investigate the susceptibility of the different species of dermatophyte strains isolated clinical specimens to five antifungal agents using the E-test method. A total of 66 specimens were collected from the nails, feet, inguinal region, trunk and hands. These strains tested MIC endpoints of E-test for amphotericin B, fluconazole, itraconazole, caspofungin, and ketoconazole were read after 72, and 96 hours incubation for each strain on RPMI 1640 agar. The dermatophytes tested included Trichophyton rubrum 43 (65.1%), Trichophyton mentagrophytes 7 (10.7%), Microsporum canis 5 (7.6%), Trichophyton tonsurans 5 (7.6%), Epidermophyton floccosum 4 (6.0%) and Trichophyton violaceum 2 (3.0%). The most active agent against all dermatophytes species was caspofungin with a minimal inhibitory concentration (MIC) range (μg/mL(-1)) (0.02-3, 0.032-4, 0.125-0.50, 0.032-2, 0.25-0.50, 0.125-0.50) and it raconazole with an MIC range (μg/mL(-1)) (0.038-1.5, 0.094-1.5, 1-32, 0.016-0.50, 0.25-0.50, 0.125-0.50). The least active agent was fluconazole with an MIC range (μg/mL(-1)) (0, 19-48, 2-256, 2-8, 256, 256, 8-24). E-test seems to be an alternative method to MIC-determination of antifungal drugs for dermatophytes species, since it is a less-laborious methodology and results could be obtained faster.

Beta-lactamic Resistance Profiles in Porphyromonas, Prevotella, and Parvimonas Species Isolated from Acute Endodontic Infections

IntroductionSusceptibility to beta-lactamic agents has changed among anaerobic isolates from acute endodontic infections. The aim of the present study was to determine the prevalence of the cfxA/cfxA2 gene in Prevotella spp., Porphyromonas spp., and Parviomonas micra strains and show its phenotypic expression. MethodsRoot canal samples from teeth with acute endodontic infections were collected and Porphyromonas, Prevotella, and Parvimonas micra strains were isolated and microbiologically identified with conventional culture techniques. The susceptibility of the isolates was determined by the minimum inhibitory concentration of benzylpenicillin, amoxicillin, and amoxicillin + clavulanate using the E-test method (AB BIODISK, Solna, Sweden). The presence of the cfxA/cfxA2 gene was determined through primer-specific polymerase chain reaction. The nitrocefin test was used to determine the expression of the lactamase enzyme. ResultsPrevotella disiens, Prevotella oralis, Porphyromonas gingivalis, and P. micra strains were susceptible to benzylpenicillin, amoxicillin, and amoxicillin + clavulanate. The cfxA/cfxA2 gene was detected in 2 of 29 isolates (6.9%). Simultaneous detection of the cfxA/cfxA2 gene and lactamase production was observed for 1 Prevotella buccalis strain. The gene was in 1 P. micra strain but was not expressed. Three strains were positive for lactamase production, but the cfxA/cfxA2 gene was not detected through polymerase chain reaction. ConclusionsThere is a low prevalence of the cfxA/cfxA2 gene and its expression in Porphyromonas spp., Prevotella spp., and P. micra strains isolated from acute endodontic infections. Genetic and phenotypic screening must be performed simultaneously to best describe additional mechanisms involved in lactamic resistance for strict anaerobes.

Kronik Akciğer Hastalığı Olan, Akut Alevlenme ve Pnömoni Tanısı ile Başvuran Çocuklarda Streptococcus pneumoniae Serotip Dağılımı ve Antimikrobiyal Duyarlılıkları

This study aimed to investigate serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates obtained from children with chronic respiratory diseases admitted to hospital with a diagnosis of acute exacerbations between 2008-2010 at Marmara University Hospital, Istanbul, Turkey. Sixty one S.pneumoniae strains isolated from the respiratory samples of patients were studied for erythromycin, clindamycin, tetracyline, trimethoprim-sulphametoxazole (TMP-SMX), vancomycin, levofloxacin susceptibilities by disk diffusion method; MIC values of penicillin and ceftriaxone were determined by E-test (AB Biodisk, Sweden). Results were evaluated according to the CLSI standards. The erythromycin-clindamycin double disc method was applied for the detection of macrolide resistance phenotypes. The presence of macrolide resistance genes, ermB, mef(A)/(E), ermTR were determined by PCR using specific primers for each gene. The serotypes were determined by multiplex PCR using specific primers for 40 different serotypes. According to CLSI criteria, penicillin resistance in S.pneumoniae isolates were found to be 8.2% (5/61) and intermediate resistance rate was 54% (33/61) for oral penicillin. Penicillin resistance were found to be only 1.6% (1/61) for parenteral penicillin. Resistance rates of erythromycin, clindamycin, tetracyline, TMP-SMX were detected as 55.8%, 46%, 47.5% and 67.2%; respectively. No resistance was detected to vancomycin and levofloxacin. Constitutive macrolide-lincosamide-streptogramin B (cMLSB) phenotype and M phenotype were observed in 82.4% (n= 28) and 17.6% (n= 6) of the macrolide resistant isolates, respectively. Inducible macrolide-lincosamide-streptogramin B (iMLSB) phenotype was not detected. The macrolid resistance genotypes, ermB, mef(A)/(E), were positive 50% and 14.7%; respectively. Both ermB and mef(A)/(E) genes were detected 35.3% of the macrolid resistant isolates. None of the isolates were positive for ermTR gene. The most common S.pneumoniae serotypes were determined as serotype 19F, 23F and 6, furthermore penicillin (34%, 15.7% and 18.4%, respectively) and macrolide (38.2%, 20.6% and 14.7%, respectively) resistance rates of those serotypes were found relatively high. Serotype covarage of 7-, 10-, 13-valent conjugated pneumococcal vaccines and 23-valent pneumococcal vaccine were 65%, 67%, 69%, and 78.6%, respectively. In our country, use of the vaccines with these coverage rates has been observed to be effective in children exposed to intensive use of antibiotics with chronic lung disease.

Phenotypic detection of Extended Spectrum Beta-Lactamases and Amp C Beta- Lactamases among nosocomial isolates in a tertiary care hospital

Background &amp; objectives: Extended-Spectrum Beta-Lactamases (ESBLs) is an important resistance mechanism in Enterobacteriaceae infections. Lack of standard guidelines from Clinical Laboratory Standards Institute (CLSI) for Amp C beta-lactamase detection poses a problem. This study was undertaken to detect ESBLs by phenotypic tests and Amp C beta-lactamase by inhibitor based method. Material and Methods: 200 consecutive non-repetitive isolates of E.coli, Klebsiella and Proteus from clinical samples were screened for ESBLs as per CLSI guidelines and confirmed by PCDT, DDST and E-tests (AB Biodisk, Biomerieux). Amp C beta lactamases were screened by cefoxitin resistance and confirmed by inhibitor (Cloxacillin) based method. Simultaneous occurrence of Amp C and ESBLs was detected by combined disk test (Neo-Sensitabs and Diatabs). Descriptive and Kappa statistics were used. Results: Out of 200 isolates studied, 131 were initially screened as ESBL producers and later 114 (57%) were confirmed by phenotypic methods. E-Test was found most sensitive phenotypic test as compared to PCDT and DDST. 13 strains resistant to cefoxitin (30?g) were found to be pure Amp C producers. Combined disk test detected 36 to be ESBL and Amp C co-producers. Surprisingly, six isolates found sensitive to cefoxitin disk were confirmed as Amp C producers by cloxacillin disk inhibition test. Conclusion: 57% ESBLs and 27.5% Amp C producers were isolated from nosocomial pathogens showing significant resistance to 3rd generation cephalosporins. Phenotypic confirmation by E-test, PCDT &amp; DDST were useful for ESBL identification and for detection of Amp C, cloxacillin was found to be an effective inhibitor. DOI: http://dx.doi.org/10.3329/bjms.v12i4.13309 Bangladesh Journal of Medical Science Vol. 12 No. 04 October ’13 Page 378-384

In vitro susceptibility of chloramphenicol against methicillin-resistant Staphylococcus aureus.

To determine the in vitro susceptibility of chloramphenicol against methicillin-resistant Staphylococcus aureus. Cross-sectional study. Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January to June 2012. One hundred and seventy four isolates of methicillin-resistant Staphylococcus aureus were included in this study using cefoxitin (30 μg) disc for detection. Minimum inhibitory concentration (MIC) of chloramphenicol against MRSA was determined by using E-strip (AB BIO DISK). The susceptibility was determined by swabbing the Mueller-Hinton agar (MHA) plates with the resultant saline suspension of MRSA and applying E-strip of chloramphenicol from AB Biodisk Sweden and determining the MIC of chloramphenicol (in μg/ml). Clinical and Laboratory Standards Institute (CLSI) recommendations of ≤ 8 μg/ml being sensitive, 16 μg/ml as intermediate and ≥32 μg/ml as resistant were followed in interpreting the results. Out of the 174 MRSA isolates, 132 (75.86%) isolates were susceptible to chloramphenicol with MICs of ≤ 8 μg/ml, 38 (21.84%) were resistant ≥32 μg/ml while 4 (2.30%) were in intermediate range with MIC of 16μg/ml. Chloramphenicol has shown good in vitro activity against MRSA and is likely to have a key role in the treatment of MRSA infections providing us a good alternative to newer expensive antimicrobials in resource limited countries.

MLST Types of Vancomycin-Resistant Enterococcus faecium Strains Isolated from Blood Cultures

Enterococci, particularly vancomycin-resistant enterococci (VRE), are important nosocomial pathogens with limited treatment options. Enterococci have low-level resistance to penicillins and aminoglycosides and are intrinsically resistant to cephalosporins. In addition, they can acquire high-level resistance to beta-lactam antibiotics, aminoglycosides and glycopeptides. The aim of this study was to determine glycopeptide resistance mechanisms and genetic relationships of vancomycin-resistant E.faecium strains isolated from blood cultures between 2003-2009 years by molecular epidemiologic methods. A total of 38 VRE strains isolated from blood cultures were included in this study. The isolates were identified by conventional methods and Phoenix 100 BD automated system (Becton Dickinson Diagnostic Systems, USA) and confirmed by sequence analysis of 16S rRNA amplicons. Antibiotic susceptibility tests were performed by the Kirby-Bauer disk diffusion method accor-ding to the CLSI standards. MIC values of vancomycin were determined in vancomycin resistant strains by E-test (AB Biodisk, Sweden) method. Vancomycin resistance genes included vanA, vanB, vanC, and vanD were investigated by polymerase chain reaction (PCR) method. Clonal relationship between strains was determined by pulsed-field gel electrophoresis (PFGE). Sequence analysis was performed for examples selected for multilocus sequence typing (MLST) of each pulsotype and subtype. Thirty eight strains of enterococci isolated from blood cultures were defined as E.faecium by phenotypic methods and confirmed by 16S rRNA sequence analysis. Vancomycin MIC values of strains were determined as > 256 µg/ml by E test. The vanA gene was detected in all isolates. Clonal relationship of 38 isolates E.faecium carrying the vanA gene was determined by PFGE and MLST methods. PFGE detected four pulsotypes (A-D) and one sporadic isolate. Twenty nine strains belonged to A pulsotype, three strains belonged to B pulsotype, two strains belonged to C pulsotype and three strains belonged to D pulsotype. Out of 29 isolates, eight strains were type A1, nine strains were type A2, six strains were type A3, two strains were type A4 and four strains were type A5. MLST identified four different sequence types (STs). Twenty nine A pulsotype and its subtypes belonged to ST117 (76.3%), three B pulsotype belonged to ST280 (7.9%), two C pulsotype belonged to ST18 (5.2%) and three D pulsotype belonged to ST17 (7.9%). In conclusion, bloodstream infections caused by VRE in our hospital arose from a dominant strain belonged to ST117. However, presence of different pulsotypes of this strain indicated that the strain had been present in the hospital for a long time and had accumulated genetic variations. In addition, infections caused by minor pulsotypes were also detected. Therefore for prevention and control of the spread of nosocomial infections caused by VRE, it is crucial to identify resistance patterns and clonal relationship of these organisms.

A comparison of Neo-Sensitabs™ tablets and paper discs in disc diffusion antimicrobial susceptibility testing

A comparative evaluation of Neo-SensiitabsTM (Rosco, Taastrup, Denmark) and BBL paper discs (Becton Dickinson, Cockeysville, MD) according to the CLSI Performance Standards for Antimicrobial Susceptibility Testing was done. A total of 303 clinical isolates were included: 60 Enterobacteriaceae (including ESBLs isolates), 63 Pseudomonas aeruginosa, 63 Staphylococcus aureus, 33 Streptococcus pneumoniae, 51 Enterococcus faecalis and 33 Beta haemolytic streptococci. Strains were tested using the disc diffusion method, including both discs and tablets. minimal inhibitory concentrations (MICs) were determined by E test (AB Biodisk, Solna, Sweden). The results were analyzed by linear regression and Pearson's correlation coefficient. The Pearson correlation coefficient of the inhibition zone diameter, related to the discs and tablets, had a high value (0.824 to 0.998). Based on the CLSI categorization of antimicrobial susceptibility (S-I-R), agreement was found within the range of 78.79 to 100%. Overall percentage of 3.31% of minor error and 0.38% of major error was observed. All of the 12 major errors occurred when there was no intermediate category. The major errors were found within isolates with SR discrepancies, and minor errors (1,64/1,67% for Neosensitabs and BBL, respectively) within isolates with IS-IR discrepancies. Antibiotic tablet sensitivity for ESBL detection was 97.14% compared to paper discs. Neo-Sensitabs™ tablets showed a high inter-correlation with BBL paper discs which indicated the possibility of using tablets as an alternative to paper discs. Key words: Disc diffusion, paper discs, Neo-Sensitabs™ tablets.

Nosocomial Infection by Sequence Type 357 Multidrug-Resistant Acinetobacter baumannii Isolates in a Neonatal Intensive Care Unit in Daejeon, Korea

Acinetobacter baumannii is an important microorganism responsible for a number of nosocomial outbreaks, in particular, in intensive care units (ICUs). We investigated a nosocomial infection caused by multidrug-resistant (MDR) A. baumannii in a neonatal intensive care unit (NICU) in Korea. A. baumannii isolates were characterized using Etest (AB Biodisk, Sweden), two multiplex PCR assays, and multilocus sequence typing (MLST) scheme. PCR and PCR mapping experiments were performed for detecting and characterizing the determinants of antimicrobial resistance. Eight strains isolated from an NICU belonged to European (EU) clone II and revealed only one sequence type (ST), namely, ST357. All the isolates were susceptible to imipenem but were resistant to amikacin, gentamicin, ceftazidime, cefepime, and ciprofloxacin. To the best of our knowledge, this is the first report of a nosocomial infection in an NICU in Korea caused by ST357 MDR/carbapenem-susceptible A. baumannii strains. This result demonstrates that nosocomial outbreaks of MDR/carbapenem-susceptible strains as well as MDR/carbapenem-resistant isolates may occur in NICUs.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Evaluation of Colistin-Ampicillin/Sulbactam Combination Efficacy in Imipenem-Resistant Acinetobacter baumannii Strains

The increasing emergence of multi-drug resistant Acinetobacter baumannii strains as nosocomial pathogens lead to the use of antimicrobial combinations in the treatment of infections due to these bacteria. The aim of this study was to determine the MIC values of colistin and ampicillin/sulbactam and their in vitro synergistic activities by E-test in order to evaluate the effect of this combination against imipenem-resistant A.baumannii isolates. A total of 33 A.baumannii strains isolated from clinical specimens as etiologic agents of nosocomial infections and identified as imipenem-resistant were included in the study. Identification of the isolates was performed by conventional methods and their imipenem resistance was detected with BD Phoenix automated system (Becton Dickinson, USA). MIC values and in vitro synergistic activity of colistin and ampicillin/sulbactam combination were analyzed by E-test (AB Biodisk, Sweden) on Mueller-Hinton agar medium. Synergistic, additive, indifferent and antagonist effects of A.baumannii strains were evaluated by fractional inhibitory concentration (FIC) index. The combination was considered to be synergistic when the FIC index was ≤ 0.5, additive when it was 1- > 0.5 and antagonistic when ≥ 2. Of the 33 strains included in the study, 21 were resistant to colistin; 30 were resistant and 3 were moderately susceptible to ampicillin/sulbactam. MIC50 and MIC90 values and MIC range of A.baumannii strains for colistin were 8, 32 and 0.13-128 µg/ml; for ampicillin/sulbactam those values were 48, 256 and 12-256 µg/ml, respectively. According to the FIC indices, 15 strains showed synergistic, four additive, five indifferent and nine antagonistic activity to colistin and ampicillin/sulbactam combination. Among the 12 colistin-susceptible strains, nine showed antagonistic, two indifferent and one synergistic activity to the tested combination while among the 21 colistin-resistant strains 14 showed synergistic, four additive and three indifferent activity. As a result, the combination of colistin with ampicillin/sulbactam, demonstrated high synergistic activity in vitro. While the synergistic effect of this combination was more significant in colistin-resistant strains, antagonistic effect of colistin-susceptible strains was found to be notable. Therefore, colistin resistance should be primarily determined before using colistin and ampicillin/sulbactam combination in A.baumannii infections since this combination seemed to be more effective in case of colistin resistance. However, these data should be supported by further advanced clinical studies.