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Objectives: To analyse the influence of antibiotic consumption on healthcare-associated healthcare onset (HAHO) Clostridioides difficile infection (CDI) in a German university hospital setting., Methods: Monthly ward-level antibiotic consumption measured in DDD/100 patient days (pd) and CDI surveillance data from five university hospitals in the period 2017 through 2019 were analysed. Uni- and multivariable analyses were performed with generalized estimating equation models., Results: A total of 225 wards with 7347 surveillance months and 4 036 602 pd participated. With 1184 HAHO-CDI cases, there was a median incidence density of 0.17/1000 pd (IQR 0.03-0.43) across all specialties, with substantial differences among specialties. Haematology-oncology wards showed the highest median incidence density (0.67/1000 pd, IQR 0.44-1.01), followed by medical ICUs (0.45/1000 pd, IQR 0.27-0.73) and medical general wards (0.32/1000 pd, IQR 0.18-0.53). Multivariable analysis revealed carbapenem (mostly meropenem) consumption to be the only antibiotic class associated with increased HAHO-CDI incidence density. Each carbapenem DDD/100 pd administered increased the HAHO-CDI incidence density by 1.3% [incidence rate ratio (IRR) 1.013; 95% CI 1.006-1.019]. Specialty-specific analyses showed this influence only to be valid for haematological-oncological wards. Overall, factors like ward specialty (e.g. haematology-oncology ward IRR 2.961, 95% CI 2.203-3.980) or other CDI cases on ward had a stronger influence on HAHO-CDI incidence density (e.g. community-associated CDI or unknown association case in same month IRR 1.476, 95% CI 1.242-1.755) than antibiotic consumption., Conclusions: In the German university hospital setting, monthly ward-level carbapenem consumption seems to increase the HAHO-CDI incidence density predominantly on haematological-oncological wards. Furthermore, other patient-specific factors seem to be equally important to control HAHO-CDI., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates., Methods: Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating., Results: In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. blaGIM-1 and blaVIM-2 were plasmid-encoded, blaVIM-4 was chromosomally encoded, while blaNDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. blaNDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the blaVIM-2- and blaGIM-1-encoding plasmids have not been described., Conclusions: These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The old antimicrobial nitroxoline is currently repurposed for oral treatment of uncomplicated urinary tract infections (UTIs)., Objectives: To investigate the in vitro activity of nitroxoline against carbapenem-resistant Acinetobacter baumannii (CRAb)., Methods: From an international collection of previously well-characterized clinical A. baumannii isolates, 34 isolates from urinary tract sources with different carbapenem-resistance mechanisms were selected. Nitroxoline activity was analysed with broth microdilution (BMD), disc diffusion (DD) and within an in vitro biofilm model. MICs of meropenem and imipenem were assessed with BMD. Susceptibility to ciprofloxacin and trimethoprim/sulfamethoxazole was investigated using DD. Escherichia coli ATCC 25922 and A. baumannii NCTC 13304 were used for quality control., Results: All isolates were carbapenem resistant (MIC90 >32 mg/L for meropenem and imipenem) and most isolates were resistant to ciprofloxacin (33/34) and trimethoprim/sulfamethoxazole (31/34). Nitroxoline yielded MIC50/90 values of 2/2 mg/L (MIC range 1-2 mg/L) and inhibition zone diameters ranging from 20 to 26 mm. In contrast, for definite eradication of biofilm-associated CRAb in vitro, higher nitroxoline concentrations (≥16 to ≥128 mg/L) were necessary for all isolates., Conclusions: Nitroxoline showed excellent in vitro activity against a collection of CRAb despite high resistance rates to other antimicrobials for parental and oral therapy of A. baumannii UTI. Currently, nitroxoline is recommended for the treatment of uncomplicated UTI in Germany with a EUCAST breakpoint limited to uncomplicated UTI and E. coli (S ≤16 mg/L). Nitroxoline could be a promising drug for oral treatment of lower UTI caused by CRAb. More data are warranted to correlate these findings with in vivo success rates., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To determine the most common tigecycline resistance mechanisms in carbapenem-resistant Acinetobacter baumannii isolates obtained during the global Tigecycline Evaluation Surveillance Trial (TEST)., Methods: Tigecycline MICs were determined by broth microdilution. WGS was used to screen for the previously identified tigecycline resistance mechanisms, as well as mutations in resistance-nodulation-cell division (RND)-type efflux pump regulators., Results: From a total 313 isolates, 113 genetically unique tigecycline-resistant isolates were analysed. The most frequent and worldwide distributed mechanism associated with tigecycline resistance was disruption of adeN, which encodes the repressor of the RND efflux pump AdeIJK, either by IS elements or nucleotide deletions causing premature stop codons. However, mutations leading to amino acid substitutions and disruption by IS elements within the two-component regulatory system adeRS, which regulates expression of the AdeABC efflux pump, correlate with higher tigecycline MICs, but these were found less frequently and were mainly restricted to Southern European countries. Furthermore, an altered version of tviB was identified in several tigecycline-resistant isolates that did not have putative resistance mutations within RND-type regulators. The resistance determinants tet(A) and tet(X), as well as resistance mutations in putative resistance determinants trm, plsC, rrf, msbA and genes encoding 30S ribosomal proteins, were not identified in any isolate., Conclusions: The most prevalent tigecycline resistance mechanisms were caused by alterations in the regulators of RND-type efflux pumps. These data provide the basis for further characterization of regulator alterations and their contribution to increased efflux and tigecycline resistance, and also should be taken into account in drug discovery programmes to overcome the contribution of efflux pumps., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture., Methods: The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization., Results: Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical., Conclusions: Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To analyse the rectal carriage rate and the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) recovered from patients upon hospital admission., Methods: Adult patients were screened at six German university hospitals from five different federal states upon hospital admission for rectal colonization with VREfm between 2014 and 2018. Molecular characterization of VREfm was performed by WGS followed by MLST and core-genome MLST analysis., Results: Of 16350 patients recruited, 263 were colonized with VREfm, with increasing prevalence rates during the 5 year study period (from 0.8% to 2.6%). In total, 78.5% of the VREfm were vanB positive and 20.2% vanA positive, while 1.2% harboured both vanA and vanB. The predominant ST was ST117 (56.7%) followed by ST80 (15%), ST203 (10.9%), ST78 (5.7%) and ST17 (3.2%). ST117/vanB VREfm isolates formed a large cluster of 96 closely related isolates extending across all six study centres and four smaller clusters comprising 13, 5, 4 and 3 isolates each. In contrast, among the other STs inter-regional clonal relatedness was rarely observed., Conclusions: To our knowledge, this is the largest admission prevalence and molecular epidemiology study of VREfm. These data provide insight into the epidemiology of VREfm at six German university hospitals and demonstrate the remarkable inter-regional clonal expansion of the ST117/vanB VREfm clone., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To evaluate the activity of the novel broad-spectrum serine β-lactamase inhibitor durlobactam (ETX2514) combined with sulbactam against global isolates of carbapenem-resistant Acinetobacter baumannii with defined carbapenem resistance mechanisms compared with reference antimicrobials with known activity against Acinetobacter spp., Methods: The susceptibility of 246 carbapenem-resistant non-duplicate A. baumannii isolates to sulbactam/durlobactam, amikacin, colistin, imipenem/sulbactam/durlobactam, imipenem, meropenem, minocycline and sulbactam was tested using broth microdilution. Isolates were obtained from various body sites from patients in 37 countries and from six world regions between 2012 and 2016. Identification of carbapenem resistance mechanisms and assignment to A. baumannii clonal lineages was based on WGS., Results: Sulbactam/durlobactam showed excellent activity comparable to colistin but superior to amikacin, minocycline and sulbactam. The sulbactam/durlobactam MIC50/90 values were 1/4 and 2/4 mg/L and the colistin MIC50/90 values were 0.5 and 1 mg/L, respectively. Comparatively, amikacin, minocycline and sulbactam MIC50/90 values were 256/≥512, 2/16 and 16/64 mg/L, respectively., Conclusions: Sulbactam/durlobactam had excellent in vitro potency against A. baumannii isolates, including those that were resistant to imipenem/meropenem, amikacin, minocycline and colistin, compared with other compounds. Sulbactam/durlobactam has the potential to become a useful addition to the limited armamentarium of drugs that can be used to treat this problem pathogen., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To investigate whether Staphylococcus aureus bloodstream infection (SAB) patients at high risk for complications or relapse benefit from combination therapy with adjunctive rifampicin or fosfomycin., Methods: In this post hoc analysis, SAB patients with native valve infective endocarditis, osteoarticular infections or implanted foreign devices were included. The co-primary endpoints were all-cause 90 day mortality and death or SAB-related late complications within 180 days. To overcome treatment selection bias and account for its time dependence, inverse probability of treatment weights were calculated and included in marginal structural Cox proportional hazard models (MSCMs)., Results: A total of 578 patients were included in the analysis, of which 313 (54%) received combination therapy with either rifampicin (n = 242) or fosfomycin (n = 58). In the multivariable MSCM, combination therapy was associated with a better outcome, that is, a lower rate of death or SAB-related late complications within 180 days (HR 0.65, 95% CI 0.46-0.92). This beneficial effect was primarily seen in patients with implanted foreign devices, in which combination therapy was associated with a lower rate of death or SAB-related late complications within 180 days (HR 0.53, 95% CI 0.35-0.79) and a lower 90 day mortality (HR 0.57, 95% CI 0.36-0.91). Upon agent-specific stratification, we found no significant differences in outcomes between combination therapy containing rifampicin and fosfomycin; however, the number of patients in most subgroups was not large enough to draw firm conclusions., Conclusions: In patients with implanted foreign devices, combination therapy was associated with a better long-term outcome. Larger prospective studies are needed to validate these findings., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To assess the admission prevalence of third-generation cephalosporin-resistant Enterobacterales (3GCREB) and to assess whether risk factors vary by β-lactamase genotype., Methods: Adult patients were recruited within 72 h of admission to general wards of six university hospitals in 2014 and 2015. Rectal swabs were screened for 3GCREB and isolates were analysed phenotypically and genotypically. Patients were questioned on potential risk factors. Multivariable analyses were performed to identify risk factors for 3GCREB colonization and for specific β-lactamases., Results: Of 8753 patients screened, 828 were 3GCREB positive (9.5%). Eight hundred and thirteen isolates were available for genotyping. CTX-M-15 was the most common ESBL (38.0%), followed by CTX-M-1 (22.5%), CTX-M-14 (8.7%), CTX-M-27 (7.5%) and SHV-ESBL (4.4%). AmpC was found in 11.9%. Interestingly, 18 Escherichia coli isolates were AmpC positive, 12 of which (67%) contained AmpC on a gene of plasmid origin [CMY (n = 10), DHA (n = 2)]. Risk factors for 3GCREB colonization varied by genotype. Recent antibiotic exposure and prior colonization by antibiotic-resistant bacteria were risk factors for all β-lactamases except CTX-M-14 and CTX-M-27. Travel outside Europe was a risk factor for CTX-M-15 and CTX-M-27 [adjusted OR (aOR) 3.49, 95% CI 2.88-4.24 and aOR 2.73, 95% CI 1.68-4.43]. A previous stay in a long-term care facility was associated with CTX-M-14 (aOR 3.01, 95% CI 1.98-4.59). A preceding hospital stay in Germany increased the risk of CTX-M-15 (aOR 1.27, 95% CI 1.14-1.41), while a prior hospital stay in other European countries increased the risk of SHV-ESBL colonization (aOR 3.85, 95% CI 1.67-8.92)., Conclusions: The detection of different ESBL types is associated with specific risk factor sets that might represent distinct sources of colonization and ESBL-specific dissemination routes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: We assessed the efficacy and safety of an oral antimicrobial regimen for short- and long-term intestinal eradication of ESBL-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EC/KP) in immunocompromised patients., Methods: We performed a randomized (2:1), double-blind multicentre Phase II study in four haematology-oncology departments. Patients colonized with ESBL-EC/KP received a 7 day antimicrobial regimen of oral colistin (2 × 106 IU 4×/day), gentamicin (80 mg 4×/day) and fosfomycin (three administrations of 3 g every 72 h), or placebo. Faecal, throat and urine specimens were collected on day 0, 6 ± 2, 11 ± 2, 28 ± 4 and 42 ± 4 after treatment initiation, and the quantitative burden of ESBL-EC/KP, resistance genes and changes in intestinal microbiota were analysed. Clinicaltrials.gov: NCT01931592., Results: As the manufacture of colistin powder was suspended worldwide, the study was terminated prematurely. Overall, 29 (18 verum/11 placebo) out of 47 patients were enrolled. The short-term intestinal eradication was marginal at day 6 (verum group 15/18, 83.3% versus placebo 2/11, 18.2%; relative risk 4.58, 95% CI 1.29-16.33; Fisher's exact test P = 0.001) and not evident at later timepoints. Quantitative analysis showed a significant decrease of intestinal ESBL-EC/KP burden on day 6. Sustained intestinal eradication (day 28 + 42) was not achieved (verum, 38.9% versus placebo, 27.3%; P = 0.299). In the verum group, mcr-1 genes were detected in two faecal samples collected after treatment. Microbiome analysis showed a significant decrease in alpha diversity and a shift in beta diversity., Conclusions: In this prematurely terminated study of a 7 day oral antimicrobial eradication regimen, short-term ESBL-EC/KP suppression was marginal, while an altered intestinal microbiota composition was clearly apparent., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain., Methods: A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT-PCR., Results: In isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot., Conclusions: This study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates.

Objectives: Fluoroquinolone resistance (FQR) in third-generation cephalosporin-resistant Enterobacteriaceae (3GCRE) presents serious limitations to antibiotic therapy. The aim of this study was to investigate whether the FQR proportion among 3GCRE differs between community-acquired (CA) and hospital-acquired (HA) isolates., Methods: In a prospective observational study covering 2014 and 2015, we monitored the occurrence of 3GCRE in adult hospitalized patients in six German university hospitals. 3GCRE clinical isolates were subdivided into CA and HA. Multivariable analysis identified factors associated with in vitro non-susceptibility to ciprofloxacin., Results: The dataset included 5721 3GCRE isolates of which 52.9% were HA and 52.7% exhibited FQR. Interestingly, the FQR proportion was higher in CA 3GCRE than in HA 3GCRE (overall, 60.1% versus 46.2%, respectively, P < 0.001). Multivariable analysis adjusting for age confirmed community acquisition as a risk factor for FQR [adjusted rate ratio (aRR) 1.33, 95% CI 1.17-1.53]. Escherichia coli and Klebsiella spp. were associated with a much higher FQR proportion than other Enterobacteriaceae species (aRR 8.14, 95% CI 6.86-9.65 and aRR 7.62 with 95% CI 6.74-8.61, respectively)., Conclusions: The high FQR proportion observed among CA 3GCRE, particularly in E. coli and Klebsiella spp., indicates that selection pressure in the outpatient setting needs to be addressed with antibiotic stewardship and other interventions in order to limit further spread of MDR.

Objectives: To investigate the molecular epidemiology, antimicrobial susceptibility and carbapenem resistance determinants of Acinetobacter baumannii isolates from respiratory tract samples of patients diagnosed with ventilator-associated pneumonia (VAP) who were enrolled in the MagicBullet clinical trial., Methods: A. baumannii isolates were prospectively cultured from respiratory tract samples from 65 patients from 15 hospitals in Greece, Italy and Spain. Susceptibility testing was performed by broth microdilution. Carbapenem resistance determinants were identified by PCR and sequencing. Molecular epidemiology was investigated using rep-PCR (DiversiLab) and international clones (IC) were identified using our in-house database., Results: Of 65 isolates, all but two isolates (97%) were resistant to imipenem and these were always associated with an acquired carbapenemase, OXA-23 (80%), OXA-40 (4.6%), OXA-58 (1.5%) or OXA-23/58 (1.5%). Resistance to colistin was 47.7%. Twenty-two isolates were XDR, and 20 isolates were pandrug-resistant (PDR). The majority of isolates clustered with IC2 (n = 54) with one major subtype comprising isolates from 12 hospitals in the three countries, which included 19 XDR and 16 PDR isolates., Conclusions: Carbapenem resistance rates were very high in A. baumannii recovered from patients with VAP. Almost half of the isolates were colistin resistant, and 42 (64.6%) isolates were XDR or PDR. Rep-PCR confirmed IC2 is the predominant clonal lineage in Europe and suggests the presence of an epidemic XDR/PDR A. baumannii clone that has spread in Greece, Italy and Spain. These data highlight the difficulty in empirical treatment of patients with A. baumannii VAP in centres with a high prevalence of carbapenem-resistant A. baumannii., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: As part of the multicentre Antibiotic Therapy Optimisation Study, MIC values of 19 non-β-lactam agents were determined for third-generation cephalosporin-resistant Escherichia coli , Klebsiella species and Enterobacter species (3GCREB) isolates collected in German hospitals., Methods: A total of 328 E. coli , 35 Klebsiella spp. (1 Klebsiella oxytoca and 34 Klebsiella pneumoniae ) and 16 Enterobacter spp. (1 Enterobacter aerogenes and 15 Enterobacter cloacae ) isolates were submitted to broth microdilution antimicrobial susceptibility testing with the MICRONAUT system. MICs of fluoroquinolones (levofloxacin and moxifloxacin), aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin, neomycin and paromomycin), tetracyclines (tetracycline, minocycline and tigecycline), macrolides (erythromycin, clarithromycin and azithromycin) and miscellaneous agents [trimethoprim/sulfamethoxazole, chloramphenicol, nitrofurantoin, colistin and fosfomycin intravenous (iv)] were determined and reviewed against 2016 EUCAST breakpoints., Results: The MIC of levofloxacin was >2 mg/L for 128 of 328 E. coli and 8 of 35 Klebsiella spp., but only 1 of 16 Enterobacter spp. Rates of resistance to trimethoprim/sulfamethoxazole were high (>70%), except for Enterobacter spp. Rates of resistance to colistin and fosfomycin iv were still low. About 20% of the tested isolates were resistant to chloramphenicol. Only 1 (of 328) E. coli isolate had an MIC of amikacin >16 mg/L and only 33 of 328 E. coli and 1 of 35 Klebsiella spp. had an MIC of tobramycin >4 mg/L, whereas average gentamicin MICs were in general more elevated. A tigecycline MIC >2 mg/L was only found for 1 of 16 Enterobacter spp., but in none of the E. coli or Klebsiella spp. isolates., Conclusions: Our study gives insight into previously unreported non-β-lactam MIC distributions of 3GCREB isolates., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: The objectives of this study were to study the presence of mutators in a set of Acinetobacter baumannii isolates and to explore whether there is a correlation between mutation rates and antibiotic resistance., Methods: The variation in mutation rate was evaluated for 237 clinical A. baumannii isolates by determining the frequency of their mutation to rifampicin resistance. For each isolate, the antibiotic resistance profile was determined by disc diffusion and/or Etest. Isolates were divided into susceptible, resistant and MDR groups according to their resistance to five groups of different antibiotics. A comparison between differences in mutation frequency (f) and strain-specific factors was performed., Results: Of the 237 isolates 32%, 18% and 50% were classified as susceptible, resistant and MDR, respectively. The f of rifampicin resistance varied between 2.2 × 10(-10) and 1.2 × 10(-6). Of the strains under investigation, 16% had an ≥2.5- to 166-fold higher f. The presence of mutators (definition ≥2.5-fold increase in f compared with ATCC 19606) in the MDR group (22%) was significantly higher (P < 0.05) than that in the susceptible and resistant groups (11% and 7%, respectively). Furthermore, f was significantly higher in the MDR group compared with that in the susceptible and resistant groups., Conclusions: The facts that 26 of 37 mutator isolates (70%) in the population were MDR and that there was a significantly higher general f in isolates exhibiting an MDR profile suggest that hypermutability can be of advantage for the organism in a selective environment with extensive exposure to antimicrobials., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: Bacteria of the genus Acinetobacter are increasingly being isolated in hospitals and are recognized as emerging nosocomial pathogens. Species identification is difficult and there is a need for simple molecular methods to differentiate between the species. Naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species and their detection by PCR can aid in species identification. The aim of this study was to develop a multiplex PCR to identify intrinsic blaOXA genes (i.e. bla(OXA-134-like), bla(OXA-211-like), bla(OXA-213-like), bla(OXA-214-like) and bla(OXA-228-like)) from Acinetobacter spp. for use as a tool for rapid species identification., Methods: Primers were designed to selectively amplify internal fragments of intrinsic blaOXA from Acinetobacter lwoffii/Acinetobacter schindleri (bla(OXA-134-like)), Acinetobacter johnsonii (bla(OXA-211-like)), Acinetobacter calcoaceticus (bla(OXA-213-like)), Acinetobacter haemolyticus (bla(OXA-214-like)) and Acinetobacter bereziniae (bla(OXA-228-like)). Multiplex PCR was performed in a total of 100 Acinetobacter isolates. Flanking primers were designed for each blaOXA subgroup and products were sequenced., Results: All A. lwoffii, A. schindleri, A. johnsonii, A. calcoaceticus, A. haemolyticus and A. bereziniae isolates were positive for their species-specific amplicons while other Acinetobacter species were negative. Thirty blaOXA novel variants were identified; the majority of these (21/30) were from A. calcoaceticus. ISAba11 was found upstream of bla(OXA-214) in four A. haemolyticus isolates, but was not associated with carbapenem resistance., Conclusions: This multiplex PCR specifically detected each of the five different blaOXA subgroups. Therefore, this method has the potential to aid in the identification of these species and monitor the spread of these genes into other Acinetobacter species.

Objectives: To characterize the mechanisms involved in reduced susceptibility to carbapenems in two Enterobacter cloacae clinical isolates., Methods: Two E. cloacae isolates recovered from different regions in Germany and showing reduced susceptibility to carbapenems were analysed. Susceptibility testing, conjugation, transformation assays, plasmid analysis, sequencing and molecular typing using rep-PCR were performed., Results: The two clinical isolates carried the bla(GIM-1) gene and showed resistance to ertapenem, with variable MIC values of imipenem and meropenem. The isolates were clonally unrelated. The bla(GIM-1) gene was located on self-transferable and non-typeable plasmids. Both isolates harboured distinct plasmids and integron structures containing the bla(GIM-1) gene cassette. Interestingly, one of the two plasmids was able to replicate in Pseudomonas aeruginosa, demonstrating its broad host range., Conclusions: This is the first identification in E. cloacae of the bla(GIM-1) gene, which is responsible for reduced susceptibility to carbapenems. We showed that this gene, previously identified in P. aeruginosa, was located in a different genetic background in E. cloacae. The bla(GIM-1) gene might spread quite efficiently in Enterobacteriaceae and P. aeruginosa, as it is difficult to detect and in addition is located on conjugative plasmids.

Objectives: Three clinical Acinetobacter baumannii isolates (A-C) were isolated from three separate patients during an outbreak in a hospital in Krakow, Poland. Isolate A was recovered first and was susceptible to carbapenems, whereas isolates B and C were resistant. The aim of this study was to investigate the differences in carbapenem susceptibility in these outbreak-related isolates., Methods: Clonal relatedness was determined using rep-PCR-based DiversiLab. The bla(OXA-51-like) genes and their upstream regions were sequenced. Expression of the genes encoding OXA-51-like and the three major porins CarO, OprD-like and 33-36 kDa Omp were investigated by semi-quantitative RT-PCR. Comparison of outer membrane protein (OMP) profiles was performed using SDS-PAGE. ISAba1-bla(OXA-82) was cloned into the shuttle vector pWH1266 and transferred into A. baumannii ATCC 17978., Results: The isolates were identical by rep-PCR and clustered with international clonal lineage 2. Sequencing of bla(OXA-51-like) revealed a conversion of OXA-66 (isolate A) into OXA-82 (isolates B and C). bla(OXA-82) was also associated with ISAba1. Expression analysis revealed overexpression of bla(OXA-82). There was no difference in OMP expression between the isolates. ISAba1-bla(OXA-82) conferred carbapenem resistance in ATCC 17978., Conclusions: Carbapenem resistance in outbreak-related isolates was mediated by conversion of OXA-66 into OXA-82 and its subsequent overexpression. This further highlights the genome plasticity of A. baumannii, leading to carbapenem resistance.

Objectives: To determine the activity of oritavancin against methicillin-resistant staphylococci, vancomycin-resistant enterococci (VRE) and β-haemolytic streptococci recently isolated from acute bacterial skin and skin structure infections or bacteraemia in western Europe., Methods: Forty-one centres in Spain (8), Italy (9), Germany (8), France (8) and the UK (8) submitted 866 isolates [204 methicillin-resistant Staphylococcus aureus (MRSA), 177 methicillin-resistant coagulase-negative staphylococci (MRCoNS), 101 VRE, 193 Streptococcus agalactiae and 191 Streptococcus pyogenes] that were collected during the first 6 months of 2011. These were re-identified and susceptibilities to oritavancin and comparators were determined., Results: Oritavancin was very active against MRSA (MIC(50)/MIC(90) 0.03/0.06 mg/L), MRCoNS (0.06/0.12 mg/L), VRE (0.03/0.06 mg/L), S. agalactiae (0.03/0.06 mg/L) and S. pyogenes (0.06/0.25 mg/L). The highest oritavancin MIC observed was 0.25 mg/L (species were S. aureus, Staphylococcus epidermidis, Staphylococcus hominis, S. agalactiae, S. pyogenes and Enterococcus faecalis)., Conclusions: These data from recently collected Gram-positive bacteria in western Europe confirm the potent in vitro activity of oritavancin against a wide range of resistant MRSA, MRCoNS and VRE isolates, including ones resistant to newer agents.

Objectives: To investigate the genetic environment of the metallo-β-lactamase gene bla(NDM-1) in an Acinetobacter baumannii isolated in 2007 in a German hospital., Methods: Antimicrobial susceptibility testing was performed and resistance genes were characterized by PCR amplification and sequencing. Transferability of β-lactam resistance was tested by broth mating assays and transformation of plasmids. The genetic background of bla(NDM-1) was analysed by primer walking. Typing of the A. baumannii strain was performed by repetitive extragenic palindromic sequence-based PCR (rep-PCR) using the DiversiLab system., Results: The multidrug-resistant A. baumannii isolate harboured β-lactamase genes bla(NDM-1) and intrinsic bla(OXA-64), but without the insertion sequence ISAba1 often located upstream. Transfer of carbapenem resistance by conjugation and transformation failed. Hybridization of isolated plasmid DNA with bla(NDM) probes was not successful. Shotgun cloning of whole genomic DNA and sequence analyses revealed that bla(NDM-1) was located between two insertion elements of ISAba125. Furthermore, this bla(NDM-1)-containing transposon structure was integrated into a chromosomal gene encoding a putative A. baumannii major facilitator superfamily (MFS) metabolite/H+ symporter., Conclusions: The metallo-β-lactamase gene bla(NDM-1) in this A. baumannii strain was integrated in the chromosome on a new transposon structure composed of two copies of insertion sequence ISAba125. The variability of the genetic environment of bla(NDM-1) likely facilitates the rapid dissemination of this gene within many Gram-negative bacterial species.

Objectives: To determine the susceptibility of Gram-negative anaerobic bacteria of the family Bacteroidaceae from hospitalized patients with intra-abdominal infections (IAIs) to moxifloxacin and other antimicrobial agents with known activity against anaerobes., Methods: Four hundred and thirty anaerobic bacterial isolates of the family Bacteroidaceae obtained from patients with IAIs were collected from 32 centres in Germany in 2007. MICs were determined using microbroth dilution for the following antimicrobials: ampicillin/sulbactam; ertapenem; meropenem; levofloxacin; moxifloxacin; clindamycin; and metronidazole. EUCAST and CLSI guidelines (for moxifloxacin) were used for interpretation., Results: Overall, metronidazole exhibited the lowest resistance rates against the study isolates (four isolates, 0.9%), while the resistance rate was 4.9% for ampicillin/sulbactam, 5.3% for ertapenem and 4.9% for meropenem. Moxifloxacin showed good activity against most Bacteroides species. Resistance rates ranged between 10% and 22% for the various species except Bacteroides vulgatus, with 59% of isolates being resistant. Clindamycin had only poor activity, with 9%-56% of Bacteroides isolates being resistant., Conclusions: Resistance among Bacteroides spp. involved in IAIs to antimicrobials with known activity against anaerobes does occur and the resistance rate observed for the carbapenems is a cause of concern. These data emphasize the need not only for periodic monitoring of the susceptibility of anaerobic pathogens to guide empirical treatment but also for species identification and susceptibility testing in selected patients with severe infections involving anaerobic bacteria.

Objectives: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND-type efflux pumps but there are no data on its activity in non-fermenters like Acinetobacter., Methods: Antimicrobial susceptibility in the absence and presence of NMP and, for comparison, phenyl-arginine-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI), was tested in laboratory and mutant strains with differing intracellular dye accumulation and expression of adeB, and in clinical isolates of Acinetobacter baumannii., Results: Based on a 4-fold or greater MIC reduction, the effects of both EPIs at low concentrations (25 mg/L) were limited. PAbetaN had a highly selective action on the reduction in the MIC of rifampicin and clarithromycin. At a higher concentration of the putative EPIs (100 mg/L), NMP was more active than PAbetaN. This effect was not limited to strains with adeB overexpression, but affected the susceptibility to linezolid, chloramphenicol and tetracycline most, and was enhanced in clinical isolates with reduced fluoroquinolone susceptibility., Conclusion: NMP can partially reverse MDR in A. baumannii and differs substantially in its activity from that of PAbetaN.

Objectives: To investigate the mechanism of ciprofloxacin resistance in isolates of Acinetobacter baumannii during two hospital outbreaks and to determine the expression level of the gene encoding the AdeB efflux pump., Methods: Isolates were previously typed by PFGE and their MICs determined by broth microdilution. The gyrA and parC genes were sequenced and the adeB gene examined by real-time reverse transcription PCR (RT-PCR)., Results: Two clonal lineages were responsible for the two hospital outbreaks. In both outbreaks, ciprofloxacin susceptibility was reduced during the course of the outbreak when compared with the index isolates. Mutations in gyrA and parC were found to have occurred during the outbreak. The MICs of non-fluoroquinolone antibiotics were raised in one clonal lineage and this was associated with a >10-fold increase in mRNA transcripts for adeB., Conclusions: We have witnessed the appearance of gyrA and parC mutations during outbreaks of A. baumannii. In parallel with these mutations, we observed up-regulation of the adeB gene associated with a decrease in susceptibility to non-fluoroquinolone antibiotics. These data illustrate the propensity for A. baumannii to develop multi-drug resistance rapidly.