Increased Resistance to Carbapenems in Proteus mirabilis Mediated by Amplification of the blaVIM-1-Carrying and IS26-Associated Class 1 Integron.

Objective: The aim of the study was to decipher the mechanisms and associated genetic determinants responsible for increased carbapenem resistance among Proteus mirabilis clinical isolates. Methods: The entire genetic structure surrounding the ß-lactam resistance genes was characterized by PCR, gene walking, and DNA sequencing. Results: A series of clinical P. mirabilis isolates were consecutively recovered from different patients at the Military hospital of Sofia, Bulgaria. They showed variable levels of resistance to carbapenems. All isolates produced the same carbapenemase VIM-1 that was chromosomally encoded. We showed that increased resistance to carbapenems was related to an increased number of blaVIM-1 gene copies. Conclusion: We showed here that increased carbapenem resistance in P. mirabilis may result from increased expression of the blaVIM-1 carbapenemase gene through multiplication of its copy number.

Carbapenem-resistant Acinetobacter baumannii: Current status of the problem in four Bulgarian university hospitals (2014-2016).

OBJECTIVES: A total of 226 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates was collected during 2014-2016 from inpatients (age range 5-88 years) in four Bulgarian university hospitals (H1-H4) to assess their antimicrobial susceptibility and to explore carbapenem resistance mechanisms as well as the molecular epidemiology of the isolates. METHODS: Antimicrobial susceptibility testing, multiplex PCR, DNA sequencing and electrotransformation experiments were performed. Epidemiological typing by random amplification of polymorphic DNA (RAPD)-PCR was also performed. RESULTS: The resistance rates were as follows: imipenem, 90.7%; meropenem, 98.2%; doripenem, 100%; amikacin, 92.9%; gentamicin, 87.2%; tobramycin, 55.8%; levofloxacin, 98.2%; trimethoprim/sulfamethoxazole, 86.3%; tigecycline, 22.1%; colistin, 0%; and ampicillin/sulbactam, 41.6%. Intrinsic blaOXA-51-like genes were found in all of the isolates. The majority of the A. baumannii isolates harboured either blaOXA-23-like associated with the upstream-located ISAba1 (26.1%) or blaOXA-40/24-like (46.7%), 45 isolates (19.9%) harboured both genes, and 1 isolate harboured blaOXA-58-like surrounded by ISAba3C upstream and ISAba3 downstream. The blaOXA-58 gene was transferable by electroporation indicating its plasmid location. Epidemiological typing revealed the dissemination of nosocomial CRAB with high clonal relatedness (70% similarity threshold) belonging to six, four, three and two clusters in H1, H2, H3, and H4 hospitals, respectively. CONCLUSIONS: The A. baumannii isolates studied were problematic nosocomial pathogens. Their multidrug resistance greatly limits therapeutic options. The persistence of endemic clones comprised of OXA carbapenemase-producing multidrug-resistant A. baumannii in the monitored hospitals over a period of ca. 3 years is of concern and requires continuous detailed investigations in the future.

NDM-1 Hazard in the Balkan States: Evidence of the First Outbreak of NDM-1-Producing Klebsiella pneumoniae in Bulgaria.

New Delhi MBL (NDM) carbapenemase-producing Klebsiella pneumoniae has become one of the most concerning multidrug-resistant pathogens. The Balkan counties are considered a reservoir for the spread of such strains based on several reports documenting NDM infections after hospitalization in this region. Nevertheless, NDM-producing K. pneumoniae have been only occasionally documented from Balkans. The current study documents the first polyclonal outbreak caused by NDM-1-producing K. pneumoniae in Bulgaria. From July 2015 to April 2016, all 25 single-patient carbapenem-nonsusceptible K. pneumoniae isolates were collected. Phenotypic and molecular screening revealed that 17 produced NDM-1 carbapenemase. All NDM-1 producers harbored blaCTX-M-15, blaCMY-4, blaTEM-1, and blaOXA-2; five also harbored blaOXA-1. In all cases, blaNDM-1 was flanked upstream by ISAba125 element and downstream by bleMBL. Pulsed-field gel electrophoresis (PFGE) clustered NDM-1-positive isolates into four distinct clonal types, A to D. MLST assigned isolates of the dominant clonal type A (n = 14) to sequence type (ST) 11, while isolates of clonal types B, C, and D to ST16, ST15, and ST391, respectively. Of interest, ST11 isolates belonged to the same PFGE type as those of the recently described NDM-1 ST11 clonal outbreak in Greece. Traveling abroad or overseas hospitalization was not reported in any case, suggesting most likely intra- and interhospital dissemination. The study presents the first polyclonal outbreak of NDM-producing K. pneumoniae in the Balkans and underlines the need for larger epidemiological studies in the region to illustrate commonalities in the transmission of NDM clones and possible sources in the community.

Colistin Resistance in KPC-2- and SHV-5-Producing Klebsiella pneumoniae Clinical Isolates in Bulgaria.

BACKGROUND/AIMS: Colistin resistance is increasingly recognized among carbapenemase-producing Klebsiella pneumoniae isolates in several European regions. The current study documents the appearance of colistin resistance among KPC-2 and SHV-5-produning K. pneumoniae strains in Bulgaria. METHODS: Four colistin-resistant K. pneumoniae isolates were recovered from 2 patients hospitalized in the anesthesiology and resuscitation clinic of a tertiary care university hospital in Sofia, Bulgaria. Microbial identification and antimicrobial susceptibility testing was performed by Vitek 2 (Biomerieux, France). ß-Lactamase genes were amplified using a panel of primers for detection of all MBL-types, KPCs, plasmid-mediated AmpCs in single PCR reactions, OXA-type carbapenemases, extended-spectrum ß-lactamases (ESBLs) and TEM enzymes. The colistin-resistant mcr-1 gene was also investigated using previously described primers and conditions. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to investigate clonality. RESULTS: The 4 K. pneumoniae isolates exhibited colistin MICs >16 mg/L and showed multidrug-resistant phenotypes, remaining intermediately susceptible only to gentamicin. They were clustered into a single PFGE clonal type and MLST assigned them to sequence type 258. All isolates possessed KPC-2 carbapenemase and SHV-5 ESBL. They were negative for the plasmid-mediated colistin-resistant mcr-1 gene, possibly implying an intrinsic mechanism of resistance. CONCLUSIONS: Although colistin use in Bulgaria only started moderately during 2014, the findings of the current study notify the appearance of colistin resistance among carbapenemase-producing Klebsiella species in another European region.

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different ß-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes blaOXA-23 or blaOXA-72; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Isolation of Acinetobacter radioresistens from a clinical sample in Bulgaria.

We report on the role of Acinetobacter radioresistens in a case of pneumonia in an elderly patient and describe the challenge of correct identification of this species. A tracheobronchial culture taken from a patient in a Bulgarian hospital yielded a pure culture of Gram-negative, lactose-non-fermenting bacilli on MacConkey agar. Genus and species identification was performed by biochemical tests and sequencing of the rpoB gene. Antimicrobial susceptibility testing and screening for blaOXA-like carbapenemase genes was done using microbroth dilution and PCR and sequencing, respectively. The bacillus growing on MacConkey agar was initially identified by biochemical tests as Acinetobacter baumannii complex. Sequencing of the rpoB gene finally identified A. radioresistens. The strain harboured the carbapenemase gene blaOXA-23 without insertion sequences upstream of this gene and was susceptible to imipenem and meropenem. In conclusion, detection of A. radioresistens remains a challenge for routine laboratory diagnostics without performance of molecular identification methods. Although A. radioresistens can be a causative agent of opportunistic infections, in the present case its involvement in the development of pneumonia is doubtful.

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria

Abstract Objectives To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Methods Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. Results The overall prevalence was: esp – 44.3%, agg/asa1 – 38.4%, ace/acm – 64.3%, efaA – 85.9%, eep – 69.4%, gelE – 64.3%, hyl – 25.1%, and cylA – 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Conclusion Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study.

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria.

OBJECTIVES: To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). METHODS: Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. RESULTS: The overall prevalence was: esp - 44.3%, agg/asa1 - 38.4%, ace/acm - 64.3%, efaA - 85.9%, eep - 69.4%, gelE - 64.3%, hyl - 25.1%, and cylA - 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. CONCLUSION: Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study.

A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae.

Rapid and specific detection of extended-spectrum ß-lactamase-producing (ESBL) bacteria is crucial both for timely antibiotic therapy when treating infected patients as well as for appropriate infection control measures aimed at curbing the spread of ESBL-producing isolates. Whereas a variety of phenotypic methods are currently available for ESBL detection, they remain time consuming and sometimes difficult to interpret while being also affected by a lack of sensitivity and specificity. Considering the longer turnaround time (TAT) of susceptibility testing and culture results, DNA-based ESBL identification would be a valuable surrogate for phenotypic-based methods. Putative ESBL-positive Enterobacteriaceae isolates (n = 330) from clinical specimen were prospectively collected in Bulgaria, Romania and Democratic Republic of Congo and tested in this study. All isolates were assessed for ESBL-production by the E-test method and those giving undetermined ESBL status were re-tested using the combination disk test. A genotypic assay successively combining qPCR detection of blaCTX-M, blaTEM and blaSHV genes with a multiplex pyrosequencing of blaTEM and blaSHV genes was developed in order to detect the most common ESBL-associated TEM and SHV single nucleotides polymorphisms, irrespective of their plasmid and/or chromosomal location. This assay was applied on all Enterobacteriaceae isolates (n = 330). Phenotypic and genotypic results matched in 324/330 (98.2%). Accordingly, real-time PCR combined with multiplex pyrosequencing appears to be a reliable and easy-to-perform assay with high-throughput identification and fast TAT (~5 h).

Amplicon identification using SparsE representation of multiplex PYROsequencing signal (AdvISER-M-PYRO): application to bacterial resistance genotyping.

MOTIVATION: Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, the objective was to develop a new method for signal processing (AdvISER-M-PYRO) to automatically analyze and interpret multiplex pyrosequencing signals. In parallel, the nucleotide dispensation order was improved by developing the SENATOR ('SElecting the Nucleotide dispensATion Order') algorithm. RESULTS: In this proof-of-concept study, quintuplex pyrosequencing was applied on eight bacterial DNA and targeted genetic alterations underlying resistance to ß-lactam antibiotics. Using SENATOR-driven dispensation order, all genetic variants (31 of 31; 100%) were correctly identified with AdvISER-M-PYRO. Among nine expected negative results, there was only one false positive that was tagged with an 'unsafe' label.

Outbreak caused by NDM-1- and RmtB-producing Escherichia coli in Bulgaria.

Twelve consecutive carbapenem-resistant Escherichia coli isolates were recovered from patients (infection or colonization) hospitalized between March and September 2012 in different units at a hospital in Bulgaria. They all produced the carbapenemase NDM-1 and the extended-spectrum-ß-lactamase CTX-M-15, together with the 16S rRNA methylase RmtB, conferring high-level resistance to all aminoglycosides. All those isolates were clonally related and belonged to the same sequence type, ST101. In addition to being the first to identify NDM-producing isolates in Bulgaria, this is the very first study reporting an outbreak of NDM-1-producing E. coli in the world.

Development of a pyrosequencing assay for rapid assessment of quinolone resistance in Acinetobacter baumannii isolates.

Rapid and reliable assessment of Acinetobacter baumannii resistance to quinolones was successfully achieved through pyrosequencing of the gyrA and parC quinolone-resistance determining regions. A strong correlation was found between quinolone resistance and mutations in gyrA codon 83 and/or in the parC gene (codons 80 or 84). Absence of QRDR mutations was associated with susceptibility to quinolones.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bulgarian hospitals.

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.