Carbapenem-resistant Klebsiella pneumoniae outbreak with monoclonal spread: Evaluation of resistance genes and ceftazidime-avibactam susceptibility.

PURPOSE: The aim of this study was to investigate ceftazidime-avibactam (CAZ-AVI) susceptibility, carbapenemase genes, and clonal relationship in carbapenem-resistant Klebsiella pneumoniae (CrKp) isolates. METHODS: A total of 28 non-repetitive CrKp isolates with positive carbapenemase production determined by the modified carbapenem inactivation method (mCIM), were included in the study. Identification of the isolates was performed with MALDI-TOF MS (VITEK-MS, bioMerieux, France). The automated system (VITEK-2, bioMerieux) and gradient diffusion test (Etest, bioMerieux) were used to determine antibiotic susceptibility. The mCIM was performed according to CLSI (2021) recommendations. CAZ-AVI susceptibility was carried out using the standard disc diffusion method. Results were evaluated according to EUCAST 2022 criteria. The blaOXA-48, blaNDM, blaKPC, blaIMP and blaVIM genes were investigated by multiplex PCR. The clonal relationship between isolates was determined by both AP-PCR and PFGE methods. RESULTS: Of the total 28 isolates, 89.3% were susceptible to CAZ-AVI. blaOXA-48 gene was found in 85.7% of the isolates, blaOXA-48+blaNDM gene in 10.7%, and blaNDM gene in 3.6%. blaKPC, blaIMP and blaVIM genes were not detected. Three clusters with three different genotypes were determined by the PFGE method. The largest cluster was Genotype A (n:24), followed by Genotype B (n:3), and Genotype C (n:1). AP-PCR was highly compatible with PFGE. The isolates of Genotype A, mostly from the intensive care unit (ICU), were evaluated as outbreak strains with monoclonal dissemination. CONCLUSIONS: OXA-48 remains the most frequently detected enzyme in CrKp strains in our country. The ceftazidime-avibactam susceptibility rate of 89.3% indicates that this antibiotic is still effective against CrKp isolates. The unnoticed outbreak detected in our study revealed the severity of intra-hospital cross-contamination affecting different wards, including the ICU. Therefore, in order to limit the spread of CrKp isolates, it is of great importance to implement strict infection control measures, and molecular surveillance programs, especially in the ICU.

[A Case of Mycobacterium abscessus Identified by Suspicious Gram Staining in the Blood Culture of a Patient with Chronic Renal Failure]. / Kronik Böbrek Yetmezlikli Hastanin Kan Kültüründe Süpheli Gram Boyamasi ile Tanimlanan Mycobacterium abscessus Olgusu.

Mycobacterium abscessus (M.abscessus), which is from the group of non-tuberculosis mycobacteria and is widely found in the natural environment, has been reported with increasing frequency as the causative agent of various infections; especially in the lower respiratory tract and in immuncompromised people. In this report, a case of M.abscessus, which developed tubular adenoma, pancytopenia and sepsis on the basis of chronic renal failure (CRF) was diagnosed by suspecting the causative agent in the Gram stain examination prepared from blood culture, was presented. A 49-year-old patient with CRF, who had complaints of weight loss, weakness, and loss of appetite for the last six months, admitted to the emergency department with a 7-8-day history of severe diarrhea and fever. Besides other tests, as the white blood cell count was 1.6 x 103/µl, neutrophil count was 80.6%, hemoglobin was 9.3 g/ dl and the platelet value was 36 x 103/µl in the blood samples, the patient was first taken into internal medicine service and then to the intensive care unit with a preliminary diagnosis of hypotension and sepsis. Meropenem and teicoplanin were started with the preliminary diagnosis of peritonitis in the internal medicine service. In addition to other tests, on the fifth day of antibiotic treatment, two consecutive sets of blood cultures were taken and sent to the microbiology laboratory. A positive signal was obtained from two aerobic blood culture samples at 42 and 45 hours of incubation in the BacT/Alert device. No bacteria were observed in the Gram staining of these samples and Erhlich Ziehl Neelsen (EZN) staining was performed because the structures considered as dye residues were noted as a result of the examination. Acid-fast bacteria were observed in the EZN-stained slide examination, and a panic report was given to the clinician. The patient died shortly after the notification was made in the evening hours. On culture plates inoculated after a positive signal, at the end of two days of aerobic incubation at 37 °C, small smooth S colonies grew on chocolate and sheep blood agar. Growing bacteria were detected as positive by EZN staining and identified as M.abscessus with 99.9% confidence by MALDI-TOF MS. After the bacterium was named as M.abscessus, the isolates were sent to the tuberculosis central laboratory of Süreyyapasa Chest Diseases and Thoracic Surgery Hospital for molecular typing. After DNA extraction from the growing colonies and polymerase chain reaction (PCR), they were typed using the GenoType NTM-DR (Hain Lifescience GmbH, Germany) kit and identified as M.abscessus, consistent with the MALDITOF MS result. After the species level identification, the erm, rrl (clarithromycin, azithromycin), and rrs (kanamycin, amikacin, and gentamicin) genes were investigated in the isolate, and it was determined that the bacteria were resistant to macrolides and sensitive to aminoglycosides. In the clinic, it should be noted that, non-tuberculous mycobacteria may play a role as an agent in immunocompromised people. On the other hand, it should be considered that non-tuberculosis bacteria may be the causative agent, with gram-positive bacilli appearing as stain residues or pale staining in Gram stains made from samples of such patients. As in this case, if the agent is seen as dye residue in blood culture Gram staining samples, it may be life-saving to suspect the agent and to report the result to the clinician accurately and quickly after EZN staining.

[Evaluation of In vitro Efficacy of Meropenem/Colistin and Meropenem/Fosfomycin Combinations on Multidrug Resistant Gram-Negative Bacilli]. / Çok Ilaca Dirençli Gram-Negatif Basillerde Meropenem/Kolistin ve Meropenem/Fosfomisin Kombinasyonlarinin In vitro Etkinliginin Degerlendirilmesi.

The rate of extensively drug-resistant and pan-resistant gram-negative rods isolated as infectious agents is increasing around the world and in Türkiye. One of the important options in the treatment of these infections is the combined use of antibiotics. Therefore, the aim of this study was to investigate the in vitro effect of meropenem/colistin and meropenem/fosfomycin combinations on carbapenem-resistant gram-negative bacilli isolated as infectious agents. Escherichia coli (n= 6), Klebsiella pneumoniae (n= 10), Pseudomonas aeruginosa (n= 5), and Acinetobacter baumannii (n= 6) isolates were recovered from blood and tracheal aspirate samples of patients hospitalized in our hospital's intensive care unit were included in the study. In the first stage of the combination study, minimal inhibitory concentrations (MIC) were investigated by broth microdilution for meropenem and colistin, and agar dilution methods for fosfomycin. In the second stage of the study, synergy, partial synergy, indifference, and antagonistic effects were investigated with the checkerboard method for the meropenem/colistin combination and the agar dilution method for the meropenem/fosfomycin combination. The checkerboard results were interpreted as follows: fractional inhibitory concentration index (FICI) values ≤ 0.5 synergy, < 0.5-≤ 1 partial synergy, > 1-≤ 4 indifference and FIC values of > 4 antagonism. MIC values obtained in the study were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Of the 27 isolates studied with the broth microdilution method, 63% were found to be colistin-resistant and 37% susceptible. The MIC values of fosfomycin against Enterobacterales group bacteria were found to be in the range of 2-2048 mg/L. Two of the six E.coli isolates and nine of the 10 K.pneumoniae isolates were found to be resistant to fosfomycin (IV). The MIC values of ≥ 128 mg/L were found in all 11 non-fermentative gram-negative rods with intrinsic resistance to fosfomycin. In the combination of meropenem/ colistin, synergy and partial synergy were observed in 11 (40.7%) of 27 isolates, an indifference effect was observed in 13 (48.2%), and antagonistic effects were observed in three (11.1%) of the isolates. The synergy and partial synergy effects of this combination were 37.5% for Enterobacterales group bacteria, 50% for E.coli, and 30% for K.pneumoniae. Regarding the 11 non-fermentative gram-negative rods included in the study, 83.3% synergy and partial synergy was found in A.baumannii for the meropenem/colistin combination, while no synergy and partial synergistic effect was found in P.aeruginosa. Meropenem/fosfomycin synergy and partial synergy effects were 83.3% (5/6) for E.coli, 100% (8/8) for K.pneumoniae, 100% (6/6) for A.baumannii, and 25% (1/4) for P.aeruginosa. In all of the isolates studied, meropenem/fosfomycin combination was found to be more effective than the meropenem/colistin combination. It would be meaningful to support these data obtained in vitro with clinical efficacy results to be obtained as a result of the application of antibiotics in vivo, taking into account the pharmacokinetic and pharmacodynamic properties of the antibiotics used in this study.

Investigation of in vitro efficacy of meropenem/polymyxin B and meropenem/fosfomycin combinations against carbapenem resistant Klebsiella pneumoniae strains.

The incidence of infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasing worldwide, and very limited number of effective antibiotics are available for therapy. In our study, the in vitro efficacy of meropenem/polymyxin B and meropenem/fosfomycin combinations against CRKP strains was investigated. The efficiency of meropenem/polymyxin B and meropenem/fosfomycin combinations was tested by checkerboard microdilution and checkerboard agar dilution methods, respectively, against 21 CRKP strains containing major carbapenem resistant genes (7 blaKPC, 7 blaOXA-48 gene, and 7 blaOXA-48+ blaNDM), and seven additional CRKP strains without carbapenemase genes.Among the 28 CRKP strains, the meropenem/polymyxin B combination was synergistic in ten (35.7%), partially synergistic in 12 (42.8%), and indifferent in six (21.4%) isolates. The meropenem/fosfomycin combination was found to be synergistic in three isolates (10.7%), partially synergistic in 20 (71.4%), and indifferent in five (17.8%). In 21 strains containing carbapenem resistance genes, meropenem/polymyxin B and meropenem/fosfomycin combinations exhibited synergistic/partial synergistic effects in 15 (71.4%) and 16 (76.2%) strains, respectively, compared to 100% synergistic/partial synergistic efficiency in both combinations in seven strains free of carbapenemase genes. No antagonistic effect was detected in either combination.Regardless of presence or absence of carbapenem resistance genes, meropenem/polymyxin B and meropenem/fosfomycin combinations both demonstrated high synergistic and partial synergistic activity against 78.4% and 82.1% of CRKP strains, respectively. Also, they have no antagonistic effects and can be used successfully to prevent therapeutic failure with monotherapy, according to our in vitro studies.

Evaluation of Broth Disk Elution Method to Determine Colistin Resistance in Klebsiella pneumoniae and Escherichia coli Strains.

BACKGROUND: The reference broth microdilution (rBMD) method for the determination of colistin resistance is very laborious and time consuming, and many manual errors can occur. There are also limitations in detection of colistin heteroresistance. Therefore, alternative methods with satisfactory performance are required for routine laboratory work. In our study, the colistin broth disk elution (CBDE) method recommended by the Clinical and Laboratory Standards Institute (CLSI) for the detection of colistin resistance in routine applications was compared with rBMD. The compatibility and error rates of the method were evaluated and its usability in routine laboratory studies was examined. METHODS: Eighty-nine multidrug resistant Klebsiella pneumoniae and five Echerichia coli strains isolated from various clinical specimens were included in the study. Identification of strains and antibiotic susceptibility tests were performed with MALDI-TOF MS (bioMerieux, France) and Vitek-2 (bioMerieux) system. Minimum inhibitory concentration (MIC) was studied in 0.125 - 128 mg/L dilution range by using polystyrene microplate and colistin sulfate salt according to ISO-standard (20776-1) recommendations for the reference BMD test. The CBDE method was performed according to the CLSI recommendations. Isolates with MIC ≤ 2 mg/L were considered susceptible, while isolates with MIC > 2 mg/L were considered resistant according to EUCAST recommendations. The performance of the CBDE method was evaluated according to ISO criteria (Categorical agreement > 90%; major error and very major error rates < 3%). RESULTS: Categorical agreement for all 58 and 36 isolates found to be resistant and susceptible, respectively, by rBMD was found to be 100% with CBDE test. Since < 1 and > 4 µg/mL values could not be determined with the CBDE method, essential agreement (EA) could not be calculated. No major or very major errors were detected. CONCLUSIONS: Our results showed that the performance of the CBDE test is good when compared to the rBMD method. According to our data, we believe that the CBDE method can be used in routine laboratories for the detection of colistin resistance.

Comparison of performance of LIAISON SARS-CoV-2 antigen assay with RT-PCR during the Omicron wave.

Due to the newly emerging Omicron variant, there is a need to re-evaluate the performance of automated antigen tests. Our study aim was to evaluate the performance of the automated Liaison SARS-CoV-2 antigen assay against reverse transcriptase polymerase chain reaction (RT-PCR) in samples with Omicron variant.A prospective study was performed on 373 combined oro-nasopharyngeal samples (NPS) randomly collected from symptomatic patients. NPS were tested with Liaison SARS-CoV-2 Ag test (DiaSorin, Italy) and DS Coronex COVID-19 Multiplex RT-PCR Diagnosis Kit (DS BioTechnology, Ankara, Turkey).Of 373 samples, 124 (33.2%) were found to be RT-PCR positive and 249 (66.8%) RT-PCR negative. Taking RT-PCR as a reference, the sensitivity and specificity of the Liaison SARS-CoV-2 Ag assay were found as 84.6% (95%CI 77.3%-90%) and 100% (95%CI 98.5%-100%), respectively. For samples with a cycle threshold (Ct) value <25 (high viral load), the sensitivity increased to 100%. When antigen concentration and Ct values were compared, a strong negative correlation between antigen and Ct values was determined (P < 0.001).The Liaison antigen test met the performance criteria recommended by the WHO for samples with the Omicron variant. In addition, it showed excellent sensitivity and specificity in patients with high viral load. Therefore, Liaison antigen test can be a reliable and useful alternative in the diagnosis of SARS-CoV-2 infection, particularly in resource-constrained laboratories.

Performance of the Rapidec®Carba NP assay for the detection of different carbapenemases in Klebsiella pneumoniae and Escherichia coli strains.

PURPOSE: The spread of infections caused by Enterobacterales strains resistant to carbapenems is a global public health problem, and early detection of carbapenemases is very important to prevent their spread. The rapid detection of carbapenemase production with the new commercial assay Rapidec® Carba NP test is based on the biochemical detection of imipenem hydrolysis. Our study aims to evaluate the performance of the Rapidec® Carba NP test in OXA-48 positive isolates highly prevalent in our country and also in isolates with more than one carbapenemase gene that have an increased prevalence and to examine whether it can be used for confirmation of carbapenemase positivity in the routine laboratory. METHODS: A total of 97 strains of 94 carbapenem-resistant Klebsiella pneumoniae and three carbapenem-resistant Escherichia coli isolated from various clinical specimens were included in the study. The results of the Rapidec® Carba NP assay were compared with those obtained by the multiplex PCR test. RESULTS: The sensitivity of the Rapidec® Carba NP test was 97.8% for all carbapenemase-positive isolates. Of 90 PCR positive isolates, one OXA-48 and one OXA-48 â€‹+ â€‹NDM positive isolates were negative with Rapidec® Carba NP test. CONCLUSIONS: The positive results detected by the Rapidec® Carba NP test make an important contribution to the early detection of carbapenemase production and infection control practices. Since two carbapenemase positive isolates were found to be negative with the Rapidec® Carba NP test in our study, it was concluded that negative results of carbapenem-resistant isolates obtained with this assay should be confirmed with an additional carbapenemase detection method to exclude false-negative results.

Phenotypic detection of carbapenemase production in carbapenem-resistant isolates with the rapid carbapenemase detection method (rCDM).

INTRODUCTION: Carbapenem antibiotics are widely used for the treatment of infections caused by multidrug-resistant bacteria. As a result of this, resistance to carbapenems is gradually increasing. Identification of carbapenemase production, one of the reasons for resistance, through molecular methods is expensive and time-consuming. In the present study, it was aimed to investigate the sensitivity of the newly developed rapid carbapenemase detection method (rCDM) as compared to the gold standard molecular method and to evaluate its consistency with another phenotypical method, the modified carbapenem inactivation method (mCIM). MATERIAL AND METHODS: In our study, a total of 152 Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli) isolated from various clinical samples of which 50 were controls were included. Strain identification was done by using VITEK®MS (bioMérieux, Marcy-I'Étoile, France), carbapenem sensitivity was tested by using VITEK®2 (bioMérieux). For carbapenem-resistant isolates, carbapenem resistance genes were detected with multiplex PCR [Carbapenem and Colistin Resistance qPCR kit (Bioeksen, Istanbul, Turkey)] kit by a molecular method. All included isolates were evaluated by the rCDM and mCIM tests in order to detect carbapenemase phenotypically. The molecular method was accepted to be the gold standard and the sensitivity of rCDM was calculated. The McNemar test was applied to analyze the difference between two phenotypic tests (rCDM and mCIM) and Cohen's Kappa analysis was applied to determine consistency. RESULTS: Out of 102 carbapenem-resistant isolates, at least one of the resistance genes in the multiplex PCR panel (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-51, blaOXA23/58, blaOXA-48) was detected in 92 and blaOXA-48 was the most common (90.2%). The sensitivity of the rapid carbapenemase detection method was found to be 100%. When the results of the two phenotypic methods were compared, no statistically significant difference could be found (PMcNemar:1, Kappa coefficient:1.00). CONCLUSION: The rapid carbapenemase detection method was found to be suitable to use in routine laboratory analysis as its sensitivity was found to be high, exhibited a good performance for detection of frequent carbapenemase types in our country (Turkey), a high consistency with mCIM, and also it is an easily applied and rapid method.

Comparison of syphilis seropositivity between non-immigrant and immigrant populations in the Anatolian side of Istanbul, Turkiye: Results of five-years retrospective study.

OBJECTIVE: Our study aimed to evaluate the seropositivity for syphilis in non- immigrant and immigrant populations and compare the results regarding demographic data. METHODS: In accordance with the reverse algorithm, syphilis tests were performed between May 2014 and December 2018 in hospitals in our service zone for syphilis screening or symptomatic disease. RESULTS: A total of 135.328 non- immigrant and 6.641 immigrant were screened for syphilis. Seropositivity rates were 1.3% in the non- immigrant and 3.8% in immigrant groups (p=0.0001). There was a statistically significant difference in terms of seropositivity rates between the various age groups in the local group and immigrant groups (except 18 25 age group) (p<0.05). Syphilis seropositivity rates were found to be lower in indigenous population than immigrant groups according to the years tested (p=0.0001). The seropositivity rates were 2.4% and 3.2% among the males (p=0.025) and 0.6% and 4.0% among females (p=0.0001) in non-immigrantand immigrant groups, respectively. Whereas, 0.6% of pregnant women in the local group and 3.7% of pregnant women in immigrant groups were seropositive for syphilis (p=0.0001). Among the HIV positive group, syphilis seropositivity was only observed in the non-immigrant group with a rate of 23.0% (p=0.0001). CONCLUSION: The antibodies against syphilis were found more frequently in immigrants than non-immigrant. Among the HIV positive individuals syphilis seropositivity was only observed in the non-immigrant group.

Detection of colistin resistance among multidrug-resistant Klebsiella pneumoniae and Escherichia coli clinical isolates in Turkey.

In this study investigation of plasmid-mediated mcr 1-5 resistance genes was performed among multidrug-resistant (MDR) colistin sensitive and resistant Klebsiella pneumoniae and Escherichia coli strains isolated in our laboratory. We aimed to evaluate automated system (Vitek-2), broth microdilution (BMD) reference method and chromogenic media performance. Totally 94 MDR K. pneumoniae and six E. coli isolates were included in the study. CHROMID® Colistin R agar (COLR) (bioMerieux, France) was used to determine the colistin resistance by chromogenic method. Standard PCR amplification was performed using specific primers to screen the plasmid-mediated mcr 1-5 genes. Sixty-one isolates were resistant to colistin and 39 were susceptible with reference BMD. The essential and categorical agreement of Vitek-2 was determined as 100 and 99%. The sensitivity of COLR medium was 100%, the specificity was 97.5%. In our study mcr-1 was detected in eight isolates, while other mcr genes were not detected. Due to the high sensitivity and specificity of the COLR medium, it can be used in routine diagnostics for the detection of colistin resistance. In our study we detected 8% prevalence of mcr-1 among MDR strains however, two mcr-1 positive isolates were found sensitive to colistin by BMD.

[Evaluation of the Two Commercial Methods Based on Broth Microdilution Against Reference Microdilution Method for Klebsiella pneumoniae Isolates]. / Klebsiella pneumoniae Izolatlarinda Sivi Mikrodilüsyon Temelli Iki Ticari Sistemin Referans Mikrodilüsyon Yöntemine Göre Degerlendirilmesi.

A rapid and reliable method for antimicrobial susceptibility test of colistin is needed because of increasing numbers of multi-resistant gram negative bacterial infections and simultaneus increasing of colistin resistance. Although broth microdilution (BMD) is recommended by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) as a reference method, the use in routine laboratory practice is limited because of the difficulties in application and time-consuming characteristics. Recently, many BMD based commercial products were developed. The study was aimed to compare the results of the two commercial systems available for the detection of colistin sensitivity with the reference method. Totally 38 Klebsiella pneumoniae strains isolated from various clinical specimens between 2017-2018 were included in our study. Identification and antibiotic susceptibility tests were performed with "matrix-assisted laser desorption/ionization timeof-flight, mass spectrometry (MALDI-TOF MS)" and Vitek-2 (bioMérieux, Marcyl'Etoile, France) systems. BMD tests were performed with Sensititre (Sensititre custom plate, Thermo Fisher Scientific, UK) and Micronaut MIC-Strip (Merlin Diagnostika GmbH, Germany) kits. Commercial BMD assays containing dehydrated colistin in the concentration range of 0.0625-128 mg/L were tested with 0.5 McFarland bacterial suspensions prepared according to the manufacturer recommendations. The reference BMD test was performed by following the recommendations of the International Organization for Standardization (ISO-20776-1). ATCC 25922 colistin-susceptible Escherichia coli and NCTC 13846 (mcr-1 positive) colistin-resistant E.coli strains were used as the quality control strains. According to the recommendations of EUCAST version 9.0, strains with minimum inhibitory concentration value of ≤ 2 mg/L were considered susceptible and strains > 2 mg/L as resistant. Thirty-five isolates were resistant to colistin by the reference method and three of them were susceptible. The Sensititre kit detected a very major error (2.8%) in one isolate; no major or very major errors were detected for the Micronaut kit. The essential and categorical agreement of the Sensititre and Micronaut kits with the reference method was defined as 74-76% and 97-100%, respectively. Colistin is the last agent for the treatment of the multi drug resistant severe bacterial infections so major and very major error for colistin should be considered equally serious. Although a very major error was detected by the Sensititre kit in one isolate, the categorical agreement of both commercial kits was greater than 90% when compared with the reference method. It was concluded that, commercially available, BMD based systems that do not require additional equipment and experience can be routinely used.