FALSE POSITIVE PHENOTYPIC DETECTION OF METALLO-BETA-LACTAMASES IN ACINETOBACTER BAUMANNII.

Phenotypic detection of metallo-ß-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.

COMPARISON OF TWO DIFFERENT METHODS FOR TIGECYCLINE SUSCEPTIBILITY TESTING IN ACINETOBACTER BAUMANNII.

- Tigecycline susceptibility testing (TST) presents a tremendous challenge for clinical microbiologists. Previous studies have shown that the Epsilometer test (E-test) and Vitek 2 automated system significantly overestimate the minimum inhibitory concentrations for tigecycline resistance compared to the broth microdilution method (BMM). This leads to very major errors or false susceptibility (i.e. the isolate is called susceptible when it is actually resistant). The aim of this study was to compare E-test against BMM for TST in carbapenem-resistant and carbapenem-susceptible Acinetobacter (A.) baumannii and to analyze changes in tigecycline susceptibility between two time periods (2009-2012 and 2013-2014), with BMM as the gold standard. Using the EUCAST criteria, the rate of resistance to tigecycline for the OXA-23 MBL-positive, OXA-23 MBL-negative and carbapenemase-negative strains for BMM was 54.5% (6/11), 29.4% (5/17) and 2.7% (1/37), respectively; the OXA-24/40 and OXA-58 producing organisms did not exhibit any resistance. With E-test, all OXA-23 MBL-positive organisms (11/11), 23.5% (4/17) of OXA-23 MBL-negative, and 4.1% of OXA-24/40 (3/74) strains displayed tigecycline resistance; there were no resistant strains among the OXA-58 and carbapenemase-negative isolates. Resistance emerged in the bacterial isolates from 2013 to 2014. Although tigecycline does not display cross-resistance, the highest rates of resistant A. baumannii isolates were observed among those producing VIM MBL, regardless of the testing method. These findings suggest that the commercial E-test does not provide reliable results for TST of A. baumannii. Further confirmation with the dilution method should be recommended, particularly in cases of serious infections.

[Carbapenemases of gram-negative bacteria]. / Karbapenemaze gram-negativnih bakterija.

Carbapenems are often antibiotics of last resort for the treatment of severe infections. They are stable to most beta-lactamases produced by gram-negative bacteria. However, bacterial enzymes named carbapenemases can efficiently hydrolyze carbapenems. They are produced most frequently by Enterobacteriaceae and non-fermentative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannnii. They belong to group A (KPC, SME, IMI, NMC), B (VIM, IMP, SPM, GIM, NDM, SIM, DIM, AIM) and D (OXA-23, OXA-24, OXA-48, OXA-58, OXA-143). The accurate and rapid laboratory identification of carbapenem-resistant isolates is important to prevent spread of such multidrug resistant strains and to avoid therapeutic failures. Therapeutic options are often limited because carbapenemases are encoded on mobile genetic elements which often harbour resistance genes to other groups of antibiotics. Thus, colistin is often the only therapeutic option.

Dissemination of Clinical Acinetobacter baumannii Isolate to Hospital Environment during the COVID-19 Pandemic.

The aim of this study was to find the source of Acinetobacter baumannii in the intensive care unit (ICU) after an outbreak during the coronavirus disease 2019 (COVID-19) pandemic, as there was no A. baumannii detected on usually screened susceptible surfaces. The screening of the ICU environment was done in April 2021 when eleven different samples were taken. One A. baumannii isolate was recovered from the air conditioner and was compared with four clinical A. baumannii isolates obtained from patients hospitalized in January 2021. Isolates were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), minimum inhibitory concentrations (MICs) were determined, and the multilocus sequence typing (MLST) was performed. The molecular identification of A. baumannii isolates as ST208, the presence of the same blaOXA-23 carbapenemase gene, and the same antibiotic susceptibility profile suggest that the isolate recovered from the air conditioner is the same as the isolates recovered from hospitalized patients. The environmental isolate was recovered three months later than the clinical isolates, emphasizing the ability of A. baumannii to survive on dry abiotic surfaces. The air conditioner in the clinical environment is an important but undoubtedly neglected source of A. baumannii outbreaks, hence, frequent disinfection of hospital air conditioners with appropriate disinfectants is mandatory to mitigate the circulation of A. baumannii between patients and the hospital environment.

Emergence of colistin resistance in Enterobacter aerogenes from Croatia.

A colistin-resistant Enterobacter aerogenes [study code 12264] was isolated from the tracheal aspirate of a 71-year-old male patient in the General Hospital [GH] in Pula, Croatia. The patient was previously treated in University Hospital Centre in Rijeka with colistin in order to eradicate Acinetobacter baumannii isolate, susceptible only to colistin and tigecycline. Genes encoding ESBLs [blaTEM, blaSHV, blaCTX-M, blaPER-1] were screened by PCR. The strain was shown to possess blaCTX-M-15 and blaTEM-1 genes. To asses genes possibly involved in resistance to colistin the chromosomal enconding mgrB gene and the plasmid-mediated mcr-1 and mcr-2 genes were screened as described previously. Mcr-1 and mcr-2 genes were not detected and mgrB gene presented a wild-type sequence. PCR-based Replicon typing method [PBRT] conducted on an E. aerogenes isolate, showed that the strain carried an IncN plasmid. Adaptive mechanisms such as changes of the bacterial cell outer membrane that cause porin decrease or presence of an efflux pump, due to selection pressure exerted by the therapeutic administration of colistin, could be responsible for the development of colistin resistance in our strain, as recently reported in E. aerogenes from France. Due to effective infection control measures, the colistin-resistant strain did not spread to other patients or hospital wards. This is the first report of an ESBL-producing, colistin-resistant E. aerogenes in clinically relevant samples such as endotracheal aspirate and blood culture, showing the presence of this rare resistance profile among Gram-negative bacteria.

Epidemic spread of OXA-48 beta-lactamase in Croatia.

PURPOSE: A dramatic increase in OXA-48 ß-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. METHODS: Carbapenemase and other ß-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 ß-lactamases. In addition to the ß-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, ß-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017.

Emergence of different Acinetobacter baumannii clones in a Croatian hospital and correlation with antibiotic susceptibility.

OBJECTIVES: During routine diagnostic laboratory work, the clinical microbiologist observed an increase of Acinetobacter baumannii isolates with three different carbapenem susceptibility patterns: susceptible, intermediate and resistant. Isolates belonging to the same carbapenem susceptibility phenotype exhibited identical susceptibility/resistance patterns to non-ß-lactam antibiotics. This prompted us to analyse the mechanisms of carbapenem-resistance and the molecular epidemiology of the isolates. A total of 59 A. baumannii isolates were analysed and grouped according to their susceptibility to imipenem: group 1 were susceptible (N=24), group 2 were intermediate (N=8) and group 3 were resistant (N=27) to imipenem. MATERIAL AND METHODS: PCR and sequencing was used to detect resistance genes. Genotyping of the isolates was performed by PFGE and MLST. RESULTS: Out of 27 resistant isolates, 20 harboured blaOXA-40-like and 7 blaOXA-23-like genes. ISAba1 was found upstream of blaOXA-51 and blaOXA-23 genes. PFGE genotyping demonstrated the existence of three major A. baumannii clones in GH Pula and determination of sequence groups showed that the isolates belonged to international clones commonly associated with multidrug-resistance. MLST (performed on six isolates) showed diverse population structure of isolates belonging to the same cluster, including ST 195, ST 231, ST 775 and ST 1095. CONCLUSIONS: A previous study conducted in 2009-2010 showed that reduced susceptibility to carbapenems in GH Pula was only associated with upregulation of the intrinsic OXA-51 ß-lactamase. In this study a shift to isolates with acquired oxacillinases, belonging to two major clones was reported.

Nursing Home as a Reservoir of Carbapenem-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is an increasingly common pathogen in healthcare settings globally. It is frequently resistant to multiple antimicrobial agents and there are recent reports on strains that are pandrug resistant. The aim of the study was to characterize the mechanisms of carbapenem resistance of A. baumannii strains from a nursing home in Zagreb and to genotype the strains. Antimicrobial susceptibility testing was performed by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). PCR was used to detect genes encoding carbapenemases of groups A, B, and D and extended-spectrum ß-lactamases. Genotyping of the strains was performed by rep-PCR. All strains were found to be resistant to ceftazidime, cefotaxime, ceftriaxone, cefepime, piperacillin/tazobactam, imipenem, meropenem, and ciprofloxacin. All, but one strain, were resistant to gentamicin. PCR revealed blaOXA-23 genes in 14, blaOXA-24 in 5, and blaVIM in 11 strains. All strains positive for blaVIM genes coharbored blaOXA-23 genes. The 14 strains with OXA-23 belonged to ICL II, whereas the 5 strains positive for blaOXA-24 belonged to ICL I. In contrast to hospitals where OXA-24/40-like ß-lactamases and OXA-58 were the most prevalent, OXA-23-like ß-lactamases are the dominant group in the nursing home. OXA-58-like ß-lactamase, which is the most widespread group, was not found. Acquisition of blaMBL genes in A. baumannii strains was observed. Rep-PCR identified two clones. Two strains A10 and A13 were alocated to a novel sequence type ST 637. Nursing homes can act as a source of dissemination of blaOXA and blaMBL genes in the environment and the possible influx to the hospital environment.

Antibiotic susceptibility of isolates from paediatric intensive care units in Zagreb.

AIM: Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program is a longitudinal global surveillance study to monitor in vitro data on microbial susceptibility in centers that prescribe meropenem. Results of the six years period (2002-2007) for the antimicrobial efficacy of meropenem compared to other broad-spectrum agents against Gram-negative and Gram-positive isolates collected at pediatric intensive care units of the University Hospital Center Zagreb in Croatia were reported. METHODS: A total of 110 Gram-negative and 43 Gram-positive pathogens from pediatric specimens were tested. The minimum-inhibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. RESULTS: There was no resistance to either imipenem or meropenem observed for Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. High resistance rates of K. pneumoniae to ceftazidime and gentamicin (50%) are a raising concern. Pseudomonas aeruginosa was the most resistant Gram-negative species with two (12%) of the strains resistant to meropenem, three (18%) to imipenem, 10 (47%) to gentamicin and six (35%) to piperacillin/tazobactam and ciprofloxacin. According to our results meropenem remains an appropriate antibiotic for the treatment of severe infections caused by Gram-negative bacteria in pediatric population. CONCLUSIONS: The results indicate that meropenem has excellent potency and spectrum of activity despite being prescribed for a long time for the treatment of seriously ill patients, and still appears to be a reliable option for the initial empirical therapy of serious nosocomial infections in children. However, later studies have shown the emergence of carbapenem-resistant Gram-negative bacteria after 2008.

Clonal spread of CTX-M-15-producing Klebsiella pneumoniae in a Croatian hospital.

This study was conducted to detect and analyse the presence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae associated with a nosocomial outbreak at a Croatian hospital. During 2007, 162 K. pneumoniae isolates with reduced susceptibility to third-generation cephalosporins were collected from various hospital units and patient specimens. Most of the strains were isolated from urine (61 %), followed by blood cultures (13 %), wound swabs (13 %), tracheal aspirates (5 %), intra-abdominal abscess aspirates (4 %), intravascular catheters (3 %) and cerebrospinal fluid (1 %). Medical wards were the most important source of the isolates (46 %); 21 % of the isolates originated from surgical intensive-care units. All patients had infections acquired during their stay in hospital. No community-acquired infections were reported. Sixty of these isolates were chosen for further analysis. A double-disc synergy test (DDST) was used to detect ESBLs. MICs were determined by the broth microdilution method according to CLSI guidelines. The transferability of ceftazidime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. Plasmids encoding ESBLs were extracted with the Macherey Nagel Mini kit according to the manufacturer's recommendations. The genotypes of the strains were compared by analysis of banding patterns generated by PFGE of XbaI-digested genomic DNA. ESBLs were found by DDST in all isolates. All strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam, piperacillin/tazobactam and ciprofloxacin. There was variable susceptibility/resistance to cefepime and gentamicin. No resistance to ceftazidime/clavulanate and carbapenems was observed. Only six strains transferred resistance to an Escherichia coli recipient strain, with low frequency. All isolates yielded an amplicon of 545 bp with consensus MA primers. Multiplex PCR was positive for group 1 CTX-M beta-lactamases. Sequencing of selected amplicons revealed the presence of bla(CTX-M-15), with coding regions containing identical nucleotide sequences. Similarly to isolates from India, our isolates contained the ISEcpI insertion sequence located upstream of the bla(CTX-M-15) gene, which has recently been demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. The isolates contained a large plasmid of approximately 150 kb. The isolates were assigned to five clusters (>85 % similarity), which contained subclusters. The results of this work provided insights into the molecular epidemiology of the spread of ESBLs in K. pneumoniae involved in an outbreak at a Croatian hospital. The hospital antibiotic policy resulted in ceftriaxone being the most heavily prescribed third-generation cephalosporin, which might be expected to select for cefotaximases such as CTX-M-15.