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Summary Aim To describe an Acinetobacter baumannii outbreak among preterm neonates in a neonatal intensive care unit (NICU) in Serbia. Methods A case–control study was conducted in the NICU at the Institute of Neonatology, Belgrade, Serbia. The case definition of A. baumannii bloodstream infection (BSI) was blood culture confirmation of systemic infection. Isolation, identification and susceptibility testing were performed using conventional methods. Molecular characterization of isolates included resistance gene detection, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. Outcomes and clinical and demographic data were obtained from patients' medical records. An infection prevention team was formed and infection control interventions were implemented. Findings During the outbreak period (May–July 2018), there were 13 cases of A. baumannii BSI among 82 hospitalized neonates. All A. baumannii strains were carbapenem resistant and susceptible to colistin. Molecular characterization of the isolates revealed that they harboured blaOXA66 and blaOXA72 beta-lactamases and belonged to sequence type 636, while the PFGE pattern indicated clonal spread. Lower gestational age, lower Apgar score, vaginal delivery and mechanical ventilation were risk factors for A. baumannii infection. Four patients died, eight patients were treated successfully with colistin, and one patient with sepsis and meningitis on dual ampicillin-sulbactam and colistin therapy recovered with sequelae. The outbreak was eventually controlled by reinforcement of the infection control measures based on a multi-tiered interventional approach. Conclusion This is the first description of an outbreak of BSI among preterm neonates caused by A. baumannii blaOXA66/blaOXA72/ST636 in Serbia.

The mechanistic details of the (PhBPE)Co-catalyzed asymmetric hydrogenation of enamides are investigated using computational and experimental approaches. Four mechanistic possibilities are compared: a direct Co(0)/Co(II) redox path, a metathesis pathway, a nonredox Co(II) mechanism featuring an azametallacycle, and a possible enamide−imine tautomerization pathway. The results indicate that the operative mechanism may depend on the type of enamide. Explicit solvent is found to be crucial for the stabilization of transition states and for a proper estimation of the enantiomeric excess. The combined results highlight the complexity of base-metal-catalyzed hydrogenations but do also provide guiding principles for a mechanistic understanding of these systems, where protic substrates can be expected to open up nonredox hydrogenation pathways.

The asymmetric Rh‐catalyzed hydrocarboxylation of α,β‐unsaturated carbonyl compounds was originally developed by Mikami and co‐workers but gives only moderate enantiomeric excesses. In order to understand the factors controlling the enantioselectivity and to propose novel ligands for this reaction, we have used computational and experimental methods to study the Rh‐catalyzed hydrocarboxylation with different bidentate ligands. The analysis of the C−CO2 bond formation transition states with DFT methods shows a preference for outer‐sphere CO2 insertion, where CO2 can undergo a backside or frontside reaction with the nucleophile. The two ligands that prefer a frontside reaction, StackPhos and tBu‐BOX, display an intriguing stacking interaction between CO2 and an N‐heterocyclic ring of the ligand (imidazole or oxazoline). Our experimental results support the computationally predicted low enantiomeric excesses and highlight the difficulty in developing a highly selective version of this reaction.

The insertion of carbon dioxide into metal element σ-bonds is an important elementary step in many catalytic reactions for carbon dioxide valorization. Here, the insertion of carbon dioxide into a family of group 10 alkyl complexes of the type (RPBP)M(CH3) (RPBP = B(NCH2PR2)2C6H4−; R = Cy or tBu; M = Ni or Pd) to generate κ1-acetate complexes of the form (RPBP)M{OC(O)CH3} is investigated. This involved the preparation and characterization of a number of new complexes supported by the unusual RPBP ligand, which features a central boryl donor that exerts a strong trans-influence, and the identification of a new decomposition pathway that results in C–B bond formation. In contrast to other group 10 methyl complexes supported by pincer ligands, carbon dioxide insertion into (RPBP)M(CH3) is facile and occurs at room temperature because of the high trans-influence of the boryl donor. Given the mild conditions for carbon dioxide insertion, we perform a rare kinetic study on carbon dioxide insertion into a late-transition metal alkyl species using (tBuPBP)Pd(CH3). These studies demonstrate that the Dimroth–Reichardt parameter for a solvent correlates with the rate of carbon dioxide insertion and that Lewis acids do not promote insertion. DFT calculations indicate that insertion into (tBuPBP)M(CH3) (M = Ni or Pd) proceeds via an SE2 mechanism and we compare the reaction pathway for carbon dioxide insertion into group 10 methyl complexes with insertion into group 10 hydrides. Overall, this work provides fundamental insight that will be valuable for the development of improved and new catalysts for carbon dioxide utilization.

The mechanism of the asymmetric hydrogenation of prochiral enamides by well-defined, neutral bis(phosphine) cobalt(0) and cobalt(II) precatalysts has been explored using(R,R)-iPrDuPhos ((R,R)-iPrDuPhos = (+)-1,2-bis[(2R,5R)-2,5-diisopropylphospholano]benzene) as a representative chiral bis(phosphine) ligand. A series of (R,R)-(iPrDuPhos)Co(enamide) (enamide = methyl-2-acetamidoacrylate (MAA), methyl(Z)-α-acetamidocinnamate (MAC), and methyl(Z)-acetamido(4-fluorophenyl)acrylate (4FMAC)) complexes (1-MAA, 1-MAC, and 1-4FMAC), as well as a dinuclear cobalt tetrahydride, [(R,R)-(iPrDuPhos)Co]2(μ2-H)3(H) (2), were independently synthesized, characterized, and evaluated in both stoichiometric and catalytic hydrogenation reactions. Characterization of (R,R)-(iPrDuPhos)Co(enamide) complexes by X-ray diffraction established the formation of the pro-(R) diastereomers in contrast to the (S)-alkane products obtained from the catalytic reaction. In situ monitoring of the cobalt-catalyzed hydrogenation reactions by UV–visible and freeze-quench electron paramagnetic resonance spectroscopies revealed (R,R)-(iPrDuPhos)Co(enamide) complexes as the catalyst resting state for all the three enamides studied. Variable time normalization analysis kinetic studies of the cobalt-catalyzed hydrogenation reactions in methanol established a rate law that is first order in (R,R)-(iPrDuPhos)Co(enamide) and H2 but independent of the enamide concentration. Deuterium-labeling studies, including measurement of an H2/D2 kinetic isotope effect and catalytic hydrogenations with HD, established an irreversible H2 addition step to the bound enamide. Density functional theory calculations support that this step is both rate and selectivity determining. Calculations, as well as HD-labeling studies, provide evidence for two-electron redox cycling involving cobalt(0) and cobalt(II) intermediates during the catalytic cycle. Taken together, these experiments support an unsaturated pathway for the [(R,R)-(iPrDuPhos)Co]-catalyzed hydrogenation of prochiral enamides.

The insertion of carbon dioxide into metal element σ-bonds is an important elementary step in many catalytic reactions for carbon dioxide valorization. Here, the insertion of carbon dioxide into a family of group 10 alkyl complexes of the type (

Capture of CO2 by amines is an attractive synthetic strategy for the formation of carbamates. Such reactions can be mediated by superbases, such as 1,1,3,3-tetramethylguanidine (TMG), with previous implications that zwitterionic superbase–CO2 adducts are able to actively transfer the carboxylate group to various substrates. Here we report a detailed investigation of zwitterionic TMG–CO2, including isolation, NMR behavior, reactivity, and mechanistic consequences in carboxylation of aniline-derivatives. Our computational and experimental mechanistic analysis shows that the reversible TMG–CO2 zwitterion is not a direct carboxylation agent. Instead, CO2 dissociates from TMG–CO2 before a concerted carboxylation occurs, where the role of the TMG is to deprotonate the amine as it is attacking a free CO2. This insight is significant, as it opens a rational way to design new synthesis strategies. As shown here, nucleophiles otherwise inert towards CO2 can be carboxylated, even without a CO2 atmosphere, using TMG–CO2 as a stoichiometric source of CO2. We also show that natural abundance 15N NMR is sensitive for zwitterion formation, complementing variable-temperature NMR studies.

In this mini-review, we provide an overview of recent computational studies on homogeneous transition metal-catalyzed carbon-carbon bond formation with CO2. We cover substitution and addition reactions involving different metals (mainly Ni, Rh, Cu) and substrates (alkanes, alkenes, alkynes, arenes) with focus on the mechanistic details. The CO2 insertion step is generally found to be rate-limiting. The reported transition state geometries for C-C bond formation mostly display three-membered cyclic arrangements involving the metal and the two reacting carbon atoms, except for reaction with Csp atoms, where acyclic conformations are observed. Other reported exceptions point to that an interaction of CO2 with the metal catalyst may not be essential. Several studies suggest that Lewis acid additives could help activating CO2 during C-C bond formation.

In transition metal-mediated carboxylation reactions, CO2 inserts into a metal–nucleophile bond. At the carboxylation transition state (TS), CO2 may interact with the metal (inner-sphere path) or may insert without being activated by the metal (outer-sphere path). Currently, there is no consensus as to which path prevails. In order to establish general predictions for the insertion of CO2 into metal–carbon bonds, we computationally analyze a series of experimentally reported Cu, Rh, and Pd complexes. Our focus is on carboxylation of aromatic substrates, including Csp3benzyl and Csp2aryl and alkenyl nucleophiles. We observe clear trends, where the nature of the nucleophile determines the preferred path: benzylic Csp3 nucleophiles favor outer-sphere and Csp2 systems favor inner-sphere CO2 insertion into the metal–carbon bond. An exception are Cu–benzyl bonds, where inner- and outer-sphere CO2 insertions are found to be competitive, highlighting the need to include both paths in mechanistic studies and in the rationalization of experimental results. For insertion into Pd–Csp2 bonds, we find that the metal–CO2 interactions at the TS are weak and may be beyond 3 Å for sterically congested ligands. Nonetheless, on the basis of a comparison to other TSs, we argue that the CO2 insertion into Pd–Csp2 bonds should be classified as inner-sphere.

Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae is one of the most important bacterial diseases of the pig respiratory tract. One hundred and forty eight Actinobacillus pleuropneumoniae strains isolated from pig's clinical samples were tested for their susceptibility to 11 antimicrobials by disk diffusion method. Isolates were obtained from the pathoanatomically altered pig's lung that died with signs of respiratory disease. Antimicrobial discs with following concentration were used: ampicillin 10 µg, amoxicillin 10 µg, amoxicillin/clavulanic acid 20/10 µg, cefotaxime 30 µg, gentamicin 10 µg, penicillin 10 IU, streptomycin 10 µg, tetracycline 30 µg, enrofloxacin 5µg, lincospectin 15/200 µg, florfenicol 30 µg and trimethoprim/sulfamethoxazole 1.25/23.75 µg. Isolated strains showed various susceptibility to tested antimicrobials. Seventy-five percent of isolates were resistant to one or more drugs frequently used in the treatment of pig's diseases at farms in Serbia. All 148 isolates were susceptible to cefotaxime, enrofloxacin and florfenicol, while 96% of them were susceptible to amoxicillin/clavulanic acid and lincospectin. Susceptibility of isolates to amoxicillin, gentamicin and trimethoprim/sulfamethoxazole were 91%, 77% and 64%, respectively. The highest resistance rates were determined for tetracycline and streptomycin, 34% and 31%, respectively. When compared to our previous results, we noticed a decrease in resistance to tetracyclines and trimethoprim/sulfamethoxazole. Our results indicate that A. pleuropneumoniae strains isolated from pig's clinical samples in Serbia show high susceptibility to antimicrobial agents which are most often used in the pig industry.

Accepted manuscript version of the following article: Pavlovic, L., Vaitla, J., Bayer, A. & Hopmann, K.H. (2018). Rhodium-Catalyzed Hydrocarboxylation: Mechanistic Analysis Reveals Unusual Transition State for Carbon–Carbon Bond Formation. Organometallics, 37(6), 941-948. Published version available at https://doi.org/10.1021/acs.organomet.7b00899. The mechanism of rhodium-COD-catalyzed hydrocarboxylation of styrene derivatives and α,β-unsaturated carbonyl compounds with CO2 has been investigated using density functional theory (PBE-D2/IEFPCM). The calculations support a catalytic cycle as originally proposed by Mikami and co-workers including β-hydride elimination, insertion of the unsaturated substrate into a rhodium–hydride bond, and subsequent carboxylation with CO2. The CO2 insertion step is found to be rate limiting. The calculations reveal two interesting aspects. First, during C–CO2 bond formation, the CO2 molecule interacts with neither the rhodium complex nor the organozinc additive. This appears to be in contrast to other CO2 insertion reactions, where CO2–metal interactions have been predicted. Second, the substrates show an unusual coordination mode during CO2 insertion, with the nucleophilic carbon positioned up to 3.6 Å away from rhodium. In order to understand the experimentally observed substrate preferences, we have analyzed a set of five alkenes: an α,β-unsaturated ester, an α,β-unsaturated amide, styrene, and two styrene derivatives. The computational results and additional experiments reported here indicate that the lack of activity with amides is caused by an overly high barrier for CO2 insertion and is not due to catalyst inactivation. Our experimental studies also reveal two putative side reactions, involving oxidative cleavage or dimerization of the alkene substrate. In the presence of CO2, these alternative reaction pathways are suppressed. The overall insights may be relevant for the design of future hydrocarboxylation catalysts.

Introduction. Beta-lactam antibiotics are the most commonly used antibacterial drugs. However, many bacteria have developed resistance to these antibiotics, and the most common form of resistance is the production of beta-lactamase enzymes. Many members of the Enterobacteriaceae family produce different types of these enzymes. Objective. The aim of this study was to perform phenotypic detection of production and identification of beta-lactamase type in Enterobacteriaceae isolated from different clinical specimens from patients hospitalized in the Clinical Center of Serbia. Methods. The strains of Enterobacteriaceae were collected between November 2011 and January 2012 in the laboratory of the Clinical Center of Serbia. The isolates were identified according to the standard microbiology procedures and confirmed by the Vitek2 automated system. Antimicrobial susceptibility testing was performed by the disk diffusion method, and the phenotypic detection of production and identification of betalactamases was performed according to previously described methodologies. Results. In this study, a total of 172 Enterobacteriaceae strains were isolated. Further testing was performed on 54/145 (37.2%) strains showing decreased susceptibility to beta-lactam antibiotics: 13/85 (15.3%) Escherichia coli, 31/46 (67.4%) Klebsiella pneumoniae and 10/14 (71.4%) Proteus mirabilis. Among them, 40/145 (27.6%) strains produced extended spectrum betalactamases (ESBLs), 9/145 (6.2%) - AmpC, 1/145 (0.7%) - K1 beta-lactamase and 4/145 (2.8%) - carbapenemases. Carbapenemases were predominantly detected in K. pneumoniae (75%). Conclusion. Enterobacteriaceae produce different types of betalactamases, and the most common type in our study was ESBLs. Production of carbapenemases detected in Enterobacteriaceae is also an associated problem. [Projekat Ministarstva nauke Republike Srbije, br. ON 175039]

The purpose of this study was to determine the presence of resistance to macrolide and tetracycline in -haemolytic streptococci which belong to group C (GCS) and group G (GGS), isolated from variuos clinical specimens collected at the Institute of Public Health of Serbia during the period 2006-2008. After determination of resistance in isolated streptococci to tested antibiotics, their phenotypic and genotypic characteristics were investigated. Resistance to erythromycin and tetracycline were evaluated in a total of 112 GGS and 29 GCS isolates. Resistance to erythromycin was determined in 6 (6.9%) GGS isolates and 4 of them were also resistant to tetracycline. Resistance to erythromycin was determined in 2 (5.4%) GCS isolates, but both isolates were sensitive to tetracycline. The erythromycinresistance phenotypes were determined by the double-disk test with erythromycin and clindamycin disks. All 8 isolates showed the MLSB macrolide resistance phenotype leading to macrolide, lincosamide and streptogramin B resistance. These 8 isolates were genotyped for the presence of the erm(TR), erm(B), mef(A) and tet(M) genes and transposon of the Tn916-Tn1545 family by polymerase chain reaction. The presence of erm(TR) gene was detected in 3 GGS isolates and in both GCS isolates, while the presence of erm(B) gene was detected in other 3 GGS isolates. The presence of tet(M) gene with transposon of the Tn916-Tn1545 family was detected in all 4 tetracycline-resistant GGS isolates. The results of this study indicate that continued monitoring of macrolide- and tetracycline- resistance in tested groups of streptococci in Belgrade and in Republic of Serbia is necessary.

Methicillin-resistant Staphylococcus aureus is a highly important human pathogen that is also a significant concern in veterinary medicine. Despite the high prevalence of colonization, clinical infections with methicillin-resistant Staphylococcus aureus appear to be rare in pigs. Methicillin-resistant Staphylococcus aureus was isolated from a sow with endometritis and her five piglets with dermatitis originating from a Serbian farm. Identification of the strains was done by automated system and confirmed by polymerase chain reaction for mecA and nuc genes. Detection of Staphylococcal Cassette Chromosome mec type was performed by multiplex polymerase chain reaction. Antimicrobial susceptibility testing on erythromycin, clindamycin, gentamicin, kanamycin, tobramycin, ciprofloxacin, tetracycline, trimethoprim/sulfamethoxazole and vancomycin was done by disc diffusion method. Six isolated strains from the infected sow and her piglets showed resistance only to tetracycline beside resistance to all beta-lactam antibiotics. In the tested methicillin-resistant Staphylococcus aureus isolates, Staphylococcal Cassette Chromosome mec type V was present. To our knowledge, this finding is the first documented detection of methicillin-resistant Staphylococcus aureus from pigs’ clinical samples in Serbia. The results of our study indicate the emergence of methicillin-resistant Staphylococcus aureus in a pig farm in Serbia highlighting the threat of this antibiotic-resistant microorganism as a pathogen causing both animal and human infections.

“One Health” has been defi ned as “the collaborative eff ort of multiple disciplines — working locally, nationally, and globally — to attain optimal health for people, animals and the environment”. One Health is a new initiative, but with the concept that extends back to ancient times. One Health is an interdisciplinary concept for complex health challenges from a holistic integrated perspective, more then a divided perspective based on different disciplines. Th ere is no isolation, wild animals, domestic animals, pathogens and diseases do not know of the political borders. The aim of the One Health initiative is to form unifi ed solutions applicable for the improvement of health of humans, animals and the environment. A workshop was organized for the representatives of all structures and levels of medical and veterinary services of Serbia. During four tasks, among joint working groups, the most important structure of One Health was proposed, introducing possible concept in Serbia.

In this work there was evaluated the method of detection of methicillin resistant Staphylococcus aureus (MRSA) by using two molecular and three phenotypic tests in investigation procedure of 70 strains of S.aureus isolated from animals. Recent findings of the new mecA homologue, mecALGA251, minimise the significance of mecA gene presence detection as a confirmation method of methicillin resistant Staphylococcus aureus identification. For this reason, along with multiplex PCR set of primers(165rDNK, nuc, mecA) for detection mecA gene, there was also used multiplex PCR set of primers (spa, mecA, pvl, mecALGA251) for differentiation mecALGA251 from mecA, with simultaneous detection of luk-PV and spa gene fragments. In all 70 investigated isolates there was detected the presence of specific 16 SrDNK fragment and nuc gene which encodes a thermostable S. aureus nuclease, while in 5 out of 70 S. aureus isolates, there was proven mecA gene presence using two multiplex PCR tests. In the investigated strains there was determined neither mecC (mecALGA251)gene presence, nor Panton Valentine Leukocidin encoding gene. By application cefoxitin disk-diffusion, latex-agglutination and two multiplex PCR tests, the identical results in identification 5 methicillin resistant out of 70 investigated S. aureus strains were obtained. In our investigation there was determined a complete correlation between the results of phenotypic and genotypic identification of methicillin resistant S. aureus. [Projekat Ministarstva nauke Republike Srbije, br. TR 31079]

The objective of this study was to analyze the occurrence of methicillin-resistantStaphylococcus aureus (MRSA) from cattle with subclinical mastitis in two dairy herds in the northwest of Serbia. All quarters reacting positive in the California Mastitis Test were sampled for bacteriological analysis. Nasal and vaginal swabs from MRSA positive cattle, and nasal swabs from humans working on farms were also collected. From 1026 cows, 212 (20.7%) suffered from subclinical mastitis. S. aureus was detected in milk samples from 84 (39.6%) cows with subclinical mastitis. Among them, MRSA were isolated from 5 (5.9%) cows. Three out of five positive cows harboured MRSA in their nose and 1 cow harboured MRSA in the vagina. No MRSA was found in human nasal swabs. Seven out of 10 MRSA isolates in farm A, and two MRSA isolates in farm B, were resistant to gentamicin and tobramycin and exhibited SCCmec type IV. The three other isolates in farm A were resistant to tetracycline and carried SCCmec elements of type V. To our knowledge, this is the first description of MRSA from bovine subclinical mastitis in Serbia. The results of our study herald the emergence of MRSA in dairy farms in Serbia, so continued surveillance is recommended. Key words: Methicillin-resistant Staphylococcus aureus, staphylococcal cassette chromosome mec typing, dairy cattle.

The purpose of our study was to determine the prevalence of macrolide resistance in 3,188 pharyngeal Streptococcus pyogenes isolates collected at the Institute of Public Health of Serbia during the period 2006-2008. The disk diffusion tests were used to determine the susceptibility of the isolates. Two hundred and sixteen S. pyogenes isolates (6.8%) were resistant to erythromycin, with 9 isolates coresistant to tetracycline: 181 isolates harbored mefA gene, 19 ermB gene, 11 ermA(TR) gene, 5 ermB and mefA genes, and 7 tetM gene. Although the prevalence of macrolide resistance in pharyngeal S. pyogenes isolates is low in Serbia, monitoring of the emergence of resistance is advisable.