Abdullah Kilic

Melatonin as an antibiotic: new insights into the actions of this ubiquitous molecule

Abstract:  The incidence of serious infections caused by multidrug‐resistant gram‐positive and gram‐negative bacteria has been increasing rapidly worldwide despite advances in antibacterial therapy in the last two decades. Among multidrug‐resistant gram‐positive and gram‐negative bacteria, methicillin‐resistant Staphylococcus aureus, carbapenem‐resistant Pseudomonas aeruginosa and Acinetobacter baumannii are of great importance, because they have emerged as primary nosocomial pathogens in hospital outbreaks. In this study, we investigated whether melatonin has antibacterial effects against these microorganisms in vitro. The minimum inhibitory concentration of melatonin was determined using a standard microdilution method at 24 and 48 hr. Melatonin inhibited microbial growth at both 24 and 48 hr; but results showed that melatonin had antibacterial effects against these microorganisms after 48 hr of incubation in lower doses [concentrations between 31.25 to 125 μg/mL (0.13–0.53 mm)]. Also, it was determined that melatonin has a more potent antimicrobial effect on gram‐negative microorganism. Among possible mechanisms, it is concluded that melatonin showed antibacterial effects by reducing intracellular substrates.

Antimicrobial Susceptibility Patterns and Staphylococcal Cassette Chromosome mec Types of, as Well as Panton-Valentine Leukocidin Occurrence among, Methicillin-Resistant Staphylococcus aureus Isolates from Children and Adults in Middle Tennessee

ABSTRACT Antimicrobial susceptibility patterns, Panton-Valentine leukocidin (PVL) occurrence, and staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from children and adults at Vanderbilt University Medical Center during a 12-month period were evaluated. A total of 1,315 MRSA isolates were collected, of which 748 (36.7%) were recovered from children. Among all isolates, 448 (34.1%) were SCCmec-II, and 847 (64.4%) were SCCmec-IV. More SCCmec-IV isolates were recovered from children than SCCmec-II isolates (424 [50.1%] versus 50 [11.2%]; odds ration [OR] = 7.98; P < 0.000001). The PVL gene was detected in 93.6% of SCCmec-IV isolates, in contrast to 0.2% in SCCmec-II isolates. Within SCCmec-IV isolates, a statistically higher PVL occurrence was noticed in children (98.1%) than in adults (89.1%) (OR = 6.34; P < 0.000001). Overall, SCCmec-II strains showed greater resistance than SCCmec-IV strains to clindamycin, erythromycin, levofloxacin, gentamicin, rifampin, minocycline, and trimethoprim-sulfamethoxazole. Both SCCmec-II and SCCmec-IV strains recovered from adults were more resistant to these antibiotics than those recovered from children. SCCmec-II strains were predominantly recovered from the respiratory tract, whereas SCCmec-IV strains were predominantly recovered from skin, soft tissue, abscesses, and surgical wounds. These data indicate that SCCmec-IV MRSA isolates frequently infect children in middle Tennessee and are likely to harbor the PVL gene.

StaphPlex System for Rapid and Simultaneous Identification of Antibiotic Resistance Determinants and Panton-Valentine Leukocidin Detection of Staphylococci from Positive Blood Cultures

ABSTRACT Phenotypic methods take several days for identification and antimicrobial susceptibility testing of staphylococcal isolates after gram-positive cocci in clusters (GPCC) are observed in positive blood cultures. We developed and validated a StaphPlex system that amplifies and detects 18 gene targets simultaneously in 1 reaction for species-level identification of staphylococci, detection of genes encoding Panton-Valentine leukocidin (PVL), and antimicrobial resistance determinants of staphylococci. The StaphPlex system was compared to phenotypic methods for organism identification and antimicrobial resistance detection for positive blood culture specimens in which GPCC were observed. Among a total of 360 GPCC specimens, 273 (75.8%), 37 (10.3%), 37 (10.3%), 1 (0.3%), 3 (0.8%), and 9 (2.5%) were identified by StaphPlex as coagulase-negative Staphylococcus (CoNS), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), or mixed infections of CoNS and MRSA, CoNS and MSSA, or nonstaphylococci, respectively, with an overall accuracy of 91.7%. The 277 CoNS-containing specimens were further identified to the species level as containing 203 (73.3%) Staphylococcus epidermidis isolates, 10 (3.6%) Staphylococcus haemolyticus isolates, 27 (9.7%) Staphylococcus hominis isolates, 1 (0.4%) Staphylococcus lugdunensis isolate, and 36 (13.0%) other CoNS isolates, with an overall accuracy of 80.1% compared to an API STAPH test and CDC reference identification. Numerous very major errors were noticed when detection of aacA, ermA, ermC, tetM, and tetK was used to predict in vitro antimicrobial resistance, but relatively few major errors were observed when the absence of these genes was used to predict susceptibility. The StaphPlex system demonstrated 100% sensitivity and specificity, ranging from 95.5% to 100.0% when used for staphylococcal cassette chromosome mec typing and PVL detection. StaphPlex provides simultaneous staphylococcal identification and detection of PVL and antimicrobial resistance determinants within 5 h, significantly shortening the time needed for phenotypic identification and antimicrobial susceptibility testing.

Efficacy of hyperbaric oxygen therapy and medical ozone therapy in experimental acute necrotizing pancreatitis.

Objectives: Our aims were to evaluate the efficacy of ozone therapy (OT) in an experimental rat model of acute necrotizing pancreatitis (ANP) and to compare its effects with hyperbaric oxygen (HBO) therapy in this entity. Methods: Forty Sprague-Dawley rats were divided into sham-operated, ANP, ANP + HBO, and ANP + OT groups. Acute necrotizing pancreatitis was induced by infusing 1-mL/kg 3% sodium taurocholate into the common biliopancreatic duct. Hyperbaric oxygen was administered twice daily at a 2.8-atm pressure for 90 minutes. Ozone therapy was set as daily intraperitoneal injections of 0.7-mg/kg ozone/oxygen gas mixture. Hyperbaric oxygen and OT were continued for 3 days after the induction of ANP. The surviving animals were killed at the fourth day, and their pancreases were harvested for biochemical, microbiological, and histopathologic analyses. Results: Serum amylase/lipase and neopterin levels and tissue oxidative stress parameters were similar to shams values in both the ANP + HBO and the ANP + OT groups. Histopathologic injury scores were significantly lower in the treatments groups than in the ANP group. When compared with the ANP group, the number of infected rats was significantly lesser in the ANP + HBO and the ANP + OT groups. Conclusions: Hyperbaric oxygen and OT reduce the severity and the mortality in the experimental rat model of ANP, and a greater benefit was received for OT comparing with HBO.

Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles.

We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf, nuc, and mecA genes in a single tube and had a detection limit of 10(5) CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus, 22 methicillin-susceptible S. aureus, and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf, nuc, and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.

Acinetobacter septicus sp. nov. Association with a Nosocomial Outbreak of Bacteremia in a Neonatal Intensive Care Unit

ABSTRACT Acinetobacter species other than Acinetobacter baumannii have rarely been reported to be associated with nosocomial outbreaks of bloodstream infections. Within a period of 1 week, seven Acinetobacter-like isolates were recovered from peripheral blood and catheter specimens of five patients at a neonatal intensive care unit (NICU) in a tertiary hospital in Turkey. All five patients had placement of central venous catheters and had received total parenteral nutrition before the onset of bacteremia. Two of the five patients died. Medical devices, tap water, aerators, water samples, various surfaces, intravenous fluids, and the hands of health care workers in the NICU were sampled and were culture negative for the bacterium. All seven of the isolates had identical biochemical reactions, antimicrobial susceptibility results, and pulsed-field gel electrophoresis patterns, indicating a clonal nosocomial outbreak. A panel of standard biochemical reaction profiles and three phenotypic commercial identification systems failed to identify these isolates. Phenotypically, the isolate differed from Acinetobacter ursingii by its hemolysis on sheep blood agar and its negative citrate utilization. Sequences of the full 16S rRNA gene, which contained at least three different gene copies with polymorphic sequences between nucleotide positions 70 and 206, were determined from the first recovered isolate. The complete 1,529- to 1,531-bp 16S rRNA gene sequences and partial 801-bp rpoB gene sequences had similarities of 99.5% and 97.2%, respectively, to an A. ursingii isolate. The DNA-DNA similarities of the strain against the type strain of A. ursingii were 64.7 and 68.7%, which were lower than the recommended threshold value of 70% for the definition of bacterial species. These data indicate that a novel Acinetobacter organism caused the nosocomial outbreak of bacteremia in the NICU unit. We propose the designation of Acinetobacter septicus sp. nov. for these isolates, with isolate AK001 as the type strain.

Clonal Spread of Serogroup W135 Meningococcal Disease in Turkey

ABSTRACT Six cases of Neisseria meningitidis serogroup W135 meningococcal infection have been reported in Turkey since 2003. Seven isolates recovered from four meningococcal meningitis patients and two asymptomatic carriers produced three distinct pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing and antigen gene sequencing showed that five isolates were indistinguishable from ST-11 (ET-37) serogroup W135 meningococci, which were first isolated in Saudi Arabia and were responsible for the worldwide outbreak among Hajj pilgrims and their contacts in 2000. The remaining two isolates, which had related PFGE patterns, differed from each other at only one of the genetic loci characterized but were not related to the ST-11 clonal complex. None of the six individuals recalled contact with a pilgrim or had traveled on the Hajj. These six individuals exhibited no time or place relationships to each other, except for the two asymptomatic carriers, who were soldiers and served in the same military unit. These data demonstrate that serogroup W135 meningococci with different genotypes, including the Hajj epidemic strain, are endemic in Turkey.

Comparison of the Efficacies of Antibiotic-Impregnated and Silver-Impregnated Ventricular Catheters on the Prevention of Infections

Background and Objective: Infection of the cerebrospinal fluid is a life-threatening condition which is usually treated with systemic antibiotics and continued ventricular drainage in children. The aim of this study was to analyze the antimicrobial activities of two antimicrobial-agent-impregnated ventricular catheters and to compare their efficacies on the bacterial cultures. Methods: Antibiotic-impregnated (clindamycin and rifampicin), silver-impregnated, and standard ventricular catheters were used in this study. The experiment was performed in 2 steps. In the first step, small pieces of the catheters were cut and incubated. Then, they were washed and placed in agar medium. Finally, the number of colonies was counted. In the second step, the pieces of catheters were placed on agar plates containing Staphylococcus aureus,Staphylococcus epidermidis, and Pseudomonas aeruginosa. The plates were incubated, and then, the inhibition zone for each catheter was measured. Results: An inhibition zone was observed only in the plates for antibiotic-impregnated catheters. In the other plates, no inhibition zone was detected. The number of colonies was lowest in the plate with the silver-impregnated catheter, followed by the antibiotic-impregnated and standard catheters. Conclusion: The antibiotic-impregnated catheter seems more effective for antimicrobial treatment. Although no inhibition zone was found in the plates for silver-impregnated catheters, these catheters allow the lowest bacterial colonization in agar.

Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles.

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.

The First Klebsiella pneumoniae Isolate Co-Producing OXA-48 and NDM-1 in Turkey

the test results were interpreted by using the CLSI criteria. The Modified Hodge test (MHT) was used to screen for the production of carbapenemase, and carbapenem-resistant isolates were examined by using real-time polymerase chain reaction for the expression of blaKPC, blaNDM-1, and blaOXA-48 [5, 6]. The nucleotide sequences were then analyzed by using an Applied Biosystems sequencer (ABI Prism 310 genetic analyzer; PE Applied Biosystems, Foster City, CA, USA). Multiple alignments were performed by using DNAMAN 4.1 software (Lynnon BioSoft, Quebec, Canada) for isolates producing NDM-1. Forty-nine of the 887 Enterobacteriaceae isolates (5.52%) were resistant to ≥1 of the three carbapenems (imipenem, meropenem, and ertapenem), and MHT revealed that all 49isolates were strong carbapenemase producers. No isolates harbored blaKPC, although 48 harbored blaOXA-48, and 1 K. pneumoniae isolate that was recovered from a patient’s urine sample was positive for both blaOXA-48 and blaNDM-1. This patient was a 75-yr-old woman who was living in Sanliurfa (on the southeastern border of Turkey) and was diagnosed as having chronic obstructive pulmonary disease, pneumonia, and hypotension. In 2013, she was transferred to the Pulmonary Diseases Department of a hospital in the central region of Turkey with severe shortness of breath, sluggishness, reduced consciousness, and weakness in her legs and arms. On the day after her admission, she developed severe respiratory fail