Analysis of potential mechanisms of non-carbapenemase mediated carbapenem resistance in Acinetobacter baumannii

Analysis of potential mechanisms of non-carbapenemase mediated carbapenem resistance in Acinetobacter baumannii | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Analysis of potential mechanisms of non-carbapenemase mediated carbapenem resistance in Acinetobacter baumannii Lisa-Marie Höfken , Sören G. Gatermann , Niels Pfennigwerth doi: https://doi.org/10.1101/2025.01.13.632675 Lisa-Marie Höfken 1 German National Reference Centre for Multidrug-resistant Gram-negative Bacteria, Department of Medical Microbiology, Ruhr-University Bochum, Universitätsstraße 150 , 44801 Bochum Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sören G. Gatermann 1 German National Reference Centre for Multidrug-resistant Gram-negative Bacteria, Department of Medical Microbiology, Ruhr-University Bochum, Universitätsstraße 150 , 44801 Bochum Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Niels Pfennigwerth 1 German National Reference Centre for Multidrug-resistant Gram-negative Bacteria, Department of Medical Microbiology, Ruhr-University Bochum, Universitätsstraße 150 , 44801 Bochum Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: niels.pfennigwerth{at}rub.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Objectives Acinetobacter baumannii is a Gram-negative nosocomial pathogen that plays an important role in the context of bacterial multidrug-resistance. Increasing resistance to carbapenems in particular is of high therapeutic relevance, as in this case only a few antibiotics remain for treatment. The mechanisms of carbapenem resistance in A. baumannii are mainly based upon carbapenemases and the mechanisms such as porin loss, efflux pumps and altered PBPs have been poorly studied to date. Methods The A. baumannii reference strain ATCC 17978 was artificially mutated by selection pressure with increasing meropenem concentrations until carbapenem resistance was achieved. Growth analyses were carried out with the mutants and MICs for relevant antibiotics were determined. In addition, the mutants were whole genome sequenced, and the sequences were compared with the wild type. As various mutagenesis attempts for targeted construction of these respective mutants were unfortunately not successful, the strain collection of the NRC was screened for isolates that showed carbapenem resistance without a detectable carbapenemase. These isolates were sequenced and analysed for abnormalities in PBPs and porins in comparison to the sequence of the reference strain ATCC 17978 and were compared to other strains that possessed a carbapenemase. Results In three of the resulting ATCC 17978 mutants, a mutation of PBP2 was observed (W366L). Theses mutants were carbapenem resistant and were not affected by avibactam in contrast to the wild type ATCC 17978. Growth experiments indicated a fitness loss compared to the wild type. As W366L was not found in the clinical isolates, we looked for other abnormalities in various genes associated with carbapenem resistance. Mutations were primarily found in the PBPs, with a mutation in PBP3 (A515V) occurring particularly often. Conclusion The results of this work support the prevailing thesis that PBP mutations in A. baumannii can lead to carbapenem resistance. Since there are hardly any studies on this hypothesis and for the most part only using outdated methods, these results are of particular relevance and further studies on this topic are recommended. Introduction Acinetobacter baumannii is a nonfermentative, Gram-negative, nonmotile and oxidase-negative bacillus. It can be found commonly in soil and water. Because of its environmental resistance, particularly to dryness, it has also been able to spread easily in the environment of hospitals and healthcare facilities. It is a very effective human colonizer in the hospital. It has been shown that care workers hands are frequently colonized with Acinetobacter species, resulting in further spread and nosocomial infections. 1 , 2 Acinetobacter baumannii was susceptible to most antibiotics until the 1970s, but since then it rapidly acquired many resistance determinants to a wide range of antibacterial agents. 2 This is a serious and ongoing problem in the treatment of infections. However, attempts to limit the spread of rapidly evolving antibiotic-resistant pathogens are best achieved by a detailed understanding of the causes that drive these resistance patterns. Resistance mechanisms For Acinetobacter there are already many resistance mechanisms known. Resistance to carbapenems, which are used as last resort antibiotics against Acinetobacter infections is of clear therapeutic relevance, as it leaves only polymyxins and tigecycline available for treatment in many cases. The most common reason for carbapenem resistance in Acinetobacter baumannii is the presence of a carbapenemase. In Germany, the most frequently found are OXA-23, OXA-72 and NDM-1. 3 But there are also other mechanisms, that confer resistance to carbapenems. CarO, a 29-kDa outer membrane channel protein which confers resistance to both imipenem and meropenem in Acinetobacter has been well characterised. 4 – 6 OprD is a well-studied porin frequently associated with imipenem resistance in Pseudomonas aeruginosa and a homologue of OprD was found in Acinetobacter , suggesting the same resistance mechanism. 7 Furthermore, reduced production of 37-, 44-, and 47 kDa OMPs in carbapenem-resistant isolates together with increased production of class C cephalosporinase has been shown to lead to carbapenem resistance. 8 Overexpression of AdeABC efflux pump may also confer high-level resistance to carbapenems in conjunction with carbapenem-hydrolysing oxacillinases. 9 However, little is known about the role of PBPs in carbapenem resistance in Acinetobacter to date. 10 Aim of this work The German National Reference Centre for multidrug-resistant Gram-negative bacteria (NRC) has a huge collection of over 80,000 resistant pathogens. In this collection, there are several carbapenem-resistant Acinetobacter baumannii isolates, most of them possessing a carbapenemase gene. However, a small portion of isolates are carbapenem-resistant without producing a carbapenemase and without any other known reason for carbapenem resistance. The aim of this work was to investigate possible reasons for non-carbapenemase-mediated carbapenem resistance in A. baumannii . Material/Methods Bacterial strains All isolates analysed in this study were clinical isolates from patients hospitalised in various German hospitals that were sent to the NRC for routine surveillance purposes. 75 % of the isolates originated from infections processes. Species identification was performed by using MALDI-TOF-MS (Bruker Daltonics, Billerica, MA, USA). All tested strains and information on origin, sequence type and β-lactamases are listed in Table 2 . Carbapenemase detection For phenotypic characterisation and for the detection or exclusion of carbapenemases, the Acinetobacter baumannii isolates were examined using a modified hodge test and a synergy test with EDTA, combined with PCRs on the carbapenemase enconding genes bla OXA-23 , bla OXA-24/40 , bla OXA-58 , bla NDM , followed by Sanger sequencing of PCR products. 11 – 13 MICs MICs were determined in triplicate using broth microdilution (BMD) with pre-configured microtiter plates (Bruker, Bremen, Germany) with CAMHB and evaluated according to EUCAST breakpoints. 14 Selection of spontaneous mutants of A. baumannii ATCC 17978 A. baumannii ATCC 17978 was plated on blood agar plates and incubated overnight at 37 °C. Next step was a macrodilution with meropenem with concentrations of up to 128 mg/L in 4 ml LB-media. Briefly, ATCC 17978 was dispensed in 0.1 % NaCl to a McFarland of 0.5 and each vial of the macrodilution was inoculated with 50 µl of the suspension. After incubation at 37 °C overnight at 200 rpm, cells of the vial with the highest concentration of meropenem where growth was visible were taken to inoculate the next macrodilution as described before. In some cases, these cells took two days to grow in the presence of meropenem. When the cells did not reach a higher level of meropenem after 3-5 cycles of incubation, a cryo-culture of this cells was prepared. Growth experiments Determination of relative cell fitness was done via monoculture growth curve experiments. Biomass monitoring was performed in triplicate using a Cell Growth Quantifier (CGQ; aquila biolabs GmbH, Baesweiler, Germany). Overnight cultures were inoculated in 50 mL of LB-media to an OD 600 nm of 0.05 ± 0.005. Shaking flasks were put on an optical sensor array and incubated for 24 h at 150 rpm and 37 °C. Data were evaluated using the CGQuant software (aquila biolabs GmbH, Baesweiler, Germany). Sequencing The molecular detection of carbapenemase genes was carried out by PCRs, as described previously (Primers used are shown in Table S1). 15 PCR products were purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and then Sanger-sequenced. If the PCR did not yield any results, the isolates were subjected to Whole-genome sequencing. Genomic Tips Kit (Qiagen, Hilden, Germany) was used to purify high molecular weight DNA. Libraries were created with Nextera® XT Library Prep Kit (Illumina, Eindhoven, The Netherlands). The sequencing was performed in-house on a MiSeq2 platform with 2 × 300 bp paired-end reads (Illumina, Eindhoven, The Netherlands). The resulting fastq-files were assembled using Spades version 3.13.1. An in-house gene search tool based on a database consisting of numerous genes associated with β-lactam resistance in A. baumannii received from NCBI was used for screening. Present ORFs were checked for correctness using BLAST ( https://blast.ncbi.nlm.nih.gov/ ) and were annotated using RAST (Rapid Annotations using Subsystems Technology). 16 – 18 Genetic characterisation of spontaneous mutants The WGS sequencing data of the A. baumannii ATCC 17978 and its spontaneous mutants were checked for SNPs via snippy on the Galaxy server (Galaxy Version 4.6.0+galaxy0). 19 Mutagenesis studies Several mutagenesis techniques were tried to create PBP mutations in ATCC 17978 chromosomally. Among others, the CRSIPR-Cas-9 system of Wang et al, 20 homologous recombination mediated by the RecAB recombination system, deletion of a PBP via resistance cassettes and suicide vectors were used. 21 Results Three of the subpopulations of ATCC 17978 survived up to a meropenem concentration of 32 mg/L. These three putative mutants of ATCC 17978 were subjected to whole-genome-sequencing together with the original ATCC 17978 to compare the sequences and to screen for putative mutations that increased meropenem resistance. Surprisingly, all three mutants showed only one single SNP compared to the origin ATCC 17978, which was located in the mrd A gene, resulting in the amino acid substitution W366L in PBP2. MICs of the A. baumannii ATCC 17978 PBP2 mutants were determined, and the results showed an increase in MICs of imipenem from 0.125 mg/L for the wild type and 2 to 4 mg/L for the mutants ( Table 2 ). A large increase in MICs of meropenem and ertapenem could also be seen (from 0.25 to 32 mg/L and 1 to 64 mg/L for meropenem and ertapenem, respectively). MICs for aztreonam increased by only one dilution step higher from 32 to 64 mg/L, but for the wildtype the combination with avibactam lowered the MIC to 8 mg/L, whereas avibactam had no effect on the mutants. MICs for ceftozolan-avibactam were higher in the mutants than in the wildtype and increased ≤ 0.25 to 8 mg/L. MICs for ceftazidim-avibactam, colistin and tigecyclin were not affected by the W366L mutation of PBP2. Growth experiments with the wild type A. baumannii ATCC 17978 and the selected PBP2 mutants showed that ATCC 17978 grew faster and achieved an overall higher cell density (OD 600 of 3.7) than the PBP2 mutants (OD 600 of 2.8) (figure 1). It was planned to verify the effect of the PBP2 mutations by mutagenesis studies in the sensitive reference strain, but unfortunately approaches with various mutagenesis techniques were not successful due to failing homologous recombination in ATCC 17978. Instead, all Acinetobacter baumannii isolates that were received by the NRC since 2009 that were tested meropenem-resistant but did not harbour a carbapenemase (n = 18) were tested again with phenotypical tests and PCRs to exclude any potentially overseen carbapenemases and subjected to whole genome sequencing to screen for the mutations observed in the selection experiments. A carbapenemase could not be detected in any of the following isolates: NRZ-18984, NRZ-29926, NRZ-30058, NRZ-41034, NRZ-44141, NRZ-52866, NRZ-53861, NRZ-56398, NRZ-69687 and NRZ-86385 (further information on origin and sequence type in table 1 ). The MICs of these isolates reached 0,25 to 2 mg/L for imipenem, 2 to 4 mg/L for meropenem and 8 to 16 mg/L for ertapenem ( Table 3 ). View this table: View inline View popup Download powerpoint Table 1 MICs of A. baumannii ATCC 17978 and its mutants in mg/L View this table: View inline View popup Download powerpoint Table 2 Background information about all tested isolates in this study, including origin, sequence type and β-lactamases View this table: View inline View popup Download powerpoint Table 3 MICs of Acinetobacter baumannii isolates in mg/L View this table: View inline View popup Download powerpoint Table 4 Results of the sequence comparison of selected proteins of the A. baumannii strains with and without carbapenemase in comparison to the reference strain ATCC 17978, or in the case of CarO to APU28151.1, as the reference strain has no intact CarO. (AAS=amino acids) These 11 isolates were subjected to whole genome sequencing via Illumina to check if there are also PBP mutations that were found in the in vitro experiment with ATCC 17978. 11 other Acinetobacter baumannii isolates that were carbapenem-resistant and carried a carbapenemase ( Table 1 ) were also sequenced for comparison. Isolates without a carbapenemase had the following mutations: A244T in PBP1a was found in two strains, P417Q and R228H in PBP1a in one isolate each and a disruption of mrc A after 264 amino acids due to an IS 1006 element was found in one isolate. The other 7 isolates had an identical PBP1a to ATCC 17978. Seven isolates showed an amino acid change in PBP1b, P112S, and one isolates with this amino acid change and additional T769A. One isolate had an amino acid change at another position in the protein, N321K. For PBP2 of the isolates without carbapenemase two amino acid changes occurred in only one isolate; T647L and P665A. PBP3 showed more often amino acid changes, there could be found seven times the amino acid change A515V, one-time N492T together with A578V and one-time Q514P. For PBP5/6 all isolates showed the same amino acid sequence as ATCC 17978. Eight isolates showed the PBP6b amino acid change P28S compared to ATCC 17978 and in one isolate occurred V287A. The protein MtgA showed ten times the amino acid change F18L and T49P and one-time F18L alone. The isolates with a carbapenemase had the following amino acid changes compared to ATCC 17978. Only two times a mutated PBP1a was found with the amino acid change A244T. Seven isolates had also the amino acid changes P112S in PBP1b. Two strains showed the mutations Q106L and P665A in PBP2. None of these strains had the W366L mutation. A515V in PBP3 was also found four times. For PBP5/6 all isolates showed the same amino acid sequence as ATCC 17978. For PBP6b the amino acid change P28S occurred in seven isolates and in one isolate there was the amino acid change A352V. For MtgA the isolates showed eight times F18L together with T49P and one-time additional F92L. The porins OprD, CarO and OmpA, that are typically relevant for carbapenem resistance in Acinetobacter baumannii , were also checked for these isolates. CarO was not found in ATCC 17978, so another reference sequence was used (APU28151.1). Sequence comparison of this porins with the isolates without carbapenemase resulted for OprD in eight times in no results, in one isolate there was the amino acid change A388T, in another L6W, F37S and T105P and in the last one R175H. CarO showed in two isolates 31 amino acid changes together with three insertions and one time 30 amino acid changes with five deleted amino acids. In one isolate there could only be found a protein with 86% identity to OmpA, eight isolates showed G52S and T216A in OmpA, one isolate T144N with T216A and one isolate only T216A. Results for the isolates with a carbapenemase were similar, in eight isolates it was not possible to find OprD, in two isolates there were OprD proteins with L247F and T406M and in one isolate there were three amino acid changes: L6W, T72A and T105P. CarO showed in three isolates 31 amino acid changes together with three insertions. OmpA of these isolates showed seven times G52S with T216A, two times only T216A alone, one time T144n with T216A and for one isolate 12 amino acid changes together with a deletion of three amino acids were found for OmpA. Discussion Although it has already been described in detail for many bacterial species that altered PBPs can lead to carbapenem resistance, 22 there are very few studies on this for Acinetobacter baumannii . Some of them are over 20 years old and the methodology was limited at the time. In 1991, Gehrlein and colleagues analysed the A. baumannii strain 4852/88 and some imipenem-resistant clones of the same strain. They saw an alteration in the PBPs in an SDS-PAGE, but it was not possible to say which PBPs were affected in detail. 23 12 years later, in 2003, a study was published by Fernandez-Cuenca et al ., in which various A. baumannii isolates with different resistance phenotypes were investigated using iodine-125 conjugate-labelled PBP binding assays. 24 , 25 The results were visualised by autoradiography in an SDS-PAGE and showed different patterns of PBPs. They concluded that the absence of a band which they assumed to be to PBP2, leads to carbapenem resistance. However, this conclusion is problematic as they only named the PBPs according to size, i.e. the largest PBP was PBP1 and so on, which is contradictory to PBP nomenclature in other species. In 2011, a paper was published that for the first time provided an overview of the PBPs in A. baumannii in relation to the genetic background. 26 It was reported that when compared to the PBPs of E. coli , A. baumannii has four high molecular weight (PBP1a, PBP1b, PBP2 and PBP3) and 3 low molecular weight PBPs (PBP5/6, PBP6b and PBP7/8) as well as the monofunctional MtgA (figure 2). However, the estimated sizes of the PBPs of A. baumannii ATCC 17978 do not correlate with the numbering that is used in E. coli , which significantly complicates literature reviews. PBP1a is not the largest PBP with 55.1 kDa, PBP1b is bigger with 88.2 kDa. Therefore, if in older publications PBPs were separated by SDS gels according to their size and then the PBPs were simply numbered from the largest to the smallest, this is incorrect with regard to the correlation of naming in E. coli . This must be considered when quoting specific statements on PBPs from the corresponding publications. Statements that altered PBP profiles could have an effect on carbapenem resistance are of course still valid. But it has to be considered that many carbapenemases were not yet known at that time and therefore may simply not have been found in some strains, as there was no whole genome sequencing performed to check this more precisely. In the abovementioned study, Cayo et al . also analysed several A. baumannii isolates from hospitals, with some of them being imipenem-sensitive and others resistant (in relation to the ECOFF). The PBP-encoding genes were sequenced and several mutations found, but they were present in both the sensitive and the resistant strains. However, all resistant isolates carried an OXA-24 carbapenemase or had an insertion of IS Aba1 upstream of bla OXA-51-like and/or the AmpC-encoding gene, which is known to result in carbapenem resistance. Furthermore, the altered PBPs may only lead to increased antibiotic tolerance, which does not necessarily exceed the ECOFF, so that isolates with those PBP mutations may still have been categorised as susceptible. In 2011, Vashist et al . published a study in which the PBPs of 20 ß-lactam-resistant strains were compared with the PBPs of ATCC 17978. 27 This was done by tagging the PBPs with Bocillin FL and subsequent size separation in an SDS-PAGE. Different band patterns and thus different compositions of PBPs were found, but again the previously mentioned problem of correctly labelling the PBPs based on size alone was encountered. Moreover, smaller point mutations or similar small changes cannot be recognised in an SDS gel. PBP2 from A. baumannii found to have a zinc binding site in the transpeptidase domain. 28 Zinc appears to be critical for the stability of PBP2 and to maintain a normal cell wall shape. Mutations that interfere with the zinc binding site have led to a loss of function of PBP2 and increased ß-lactam resistance. The amino acids D350, D365, H371 and C384 are probably responsible for the binding of zinc. The mutation W366L in PBP2 in our spontaneous mutants may also have these effects especially because the base exchange is directly one amino acid next to the potential zinc-binding amino acid D365. It was furthermore shown that mutations in AdeB (F136L and G280S) and PBP3, including A515V, lead to meropenem MICs of 32 mg/L and that A515V is very close to the binding site for meropenem and therefore probably influences binding. 29 In this study, the A515V mutation in PBP3 was also found frequently in A. baumannii isolates, that were carbapenem resistant without harbouring a carbapenemase. These results support the hypothesis of Hawkey et al . that this particular mutation could lead to meropenem resistance in A. baumannii . A mutagenesis study on the D,D-transpeptidases of A. baumannii and discovered that the PBP3 of A. baumannii could not be deleted and is therefore essential for the growth and secondly, mutants lacking the three PBPs 1a, 1b and 2 were up to 8 times more sensitive to ß-lactams. 30 Not much has been published on the involvement of PBPs in carbapenem resistance in Acinetobacter spp., but since PBPs do play a role in other species, it is reasonable to assume that this is also the case here. The results of this study show the potential involvement of an in vitro induced PBP2 mutation in carbapenem resistance. In some carbapenem resistant isolates from our strain collection, in which no carbapenemase could be detected, some PBP mutations were also found, mainly A515V in PBP3. As this mutation was also found in other strains with carbapenemase, further studies would be interesting to see whether carbapenem resistance is also prevalent without a carbapenemase present. Mutagenesis studies were attempted in this study, but were unfortunately unsuccessful, as A. baumannii ATCC 17978 did not allow homologous recombination, regardless of the mutagenesis technique (CRISPR-Cas9, assisted homologous recombination, suicide vectors or deletion cassettes). Sometimes initial screening gave the impression of successful cloning, but after sequencing it turned out that the relevant resistance cassettes had only been incorporated into transposons or IS elements and that no homologous recombination had taken place at the desired site. For this reason, various mutagenesis techniques were tried and varied, but all failed due to the missing homologous recombination. A slightly reduced growth of the mutants compared to the wild type strain possible indicates a loss of fitness due to the PBP2 mutation. This could explain why such mutations are rarely found in wild Acinetobacter baumannii isolates. However, there is still a risk that such a mutation could occur during carbapenem therapy and lead to treatment failure without horizontal gene transfer and the involvement of carbapenemases. In combination with some β-lactamases or porin losses or efflux pumps, PBP mutations can have some important effects on the resistance profile. The role of Omps in antibiotic resistance is very speculative because most Acinetobacter Omps haven’t even been annotated or studied yet. However, there is some research on OmpA, OprD and CarO and their role in β-lactam resistance. OmpA is the most prevalent porin in A. baumannii and disruption mutants were found to have increased susceptibility to aztreonam, chloramphenicol, imipenem, meropenem and nalidixic acid. 31 Studies also suggest that OmpA may be coupled to efflux pumps that force antimicrobial compounds out of the periplasmic space. 32 Most of the A. baumannii isolates examined in this study had only two amino acid differences compared to ATCC 17978 in OmpA (G52S, T216A). Consequently, this could simply be an allelic variation. Mutagenesis and expression studies would be required to further investigate the influence on β-lactam resistance. However, there is one strain that does not appear to have intact OmpA (NRZ-18984), which could potentially lower its resistance level against carbapenems compared to the other isolates. CarO is another A. baumannii porin associated with carbapenem susceptibility. Some studies observed a selective imipenem influx in A. baumannii , 5 while another study created a liposome model system embedded with CarO that denied the ability to transport carbapenems through CarO. 33 Despite this solitary observation, there is a wealth of evidence from different research groups pointing to the role of CarO in antibiotic resistance. 34 In this study there were several isolates with a wild-type CarO, but also some isolates with an altered form with 30 or more amino acid substitutions. This may also contribute to carbapenem resistance, independent of the presence of a carbapenemase. This study also found that the sensitive reference strain ATCC 17978 does not possess CarO, which, according to the current state of research, could lead to imipenem resistance. However, this strain is absolutely sensitive to imipenem. The loss of CarO alone does not appear to be sufficient to confer relevant resistance to imipenem. However, in combination with other resistance determinants, it may have an additive effect. OprD was first identified in studies of the outer membrane of carbapenem-resistant A. baumannii isolates. 7 It is an orthologue of a porin involved in the transport of basic amino acids and imipenem in P. aeruginosa. 35 While an isogenic deletion mutant of A. baumannii OprD did not affect the MICs of β-lactams, 32 a significant reduction in the MIC of imipenem, ertapenem and meropenem was observed in another Acinetobacter species, A. baylyi spp.. 36 Despite these contradictory reports of OprD and antibiotic resistance in A. baumannii , SNPs and insertion elements in OprD were frequently identified in MDR A. baumannii, which may indicate a role for OprD in resistance. In this study about three quarters of isolates did not pocess the oprD porin and all other isolates showed single amino acid changes in this porin. No correlation could be drawn between abnormalities in porins and the presence of a carbapenemase in the isolates examined here. Further studies are needed to break down the complex influence of porins on the resistance of Acinetobacter baumannii . Conclusion Many reviews on Acinetobacter baumannii state that carbapenem resistance is caused by PBPs, among other mechanisms. However, a closer look at the studies available to date reveals that the data on this is rather sparse and should be viewed with caution. In this study, it was found that resistance to meropenem could be induced in vitro with a sensitive reference strain, resulting from a single amino acid exchange in PBP2. In addition, further PBP mutations were found in other clinical isolates that were carbapenem-resistant but do not possess a carbapenemase, further underlining the potential importance of PBP mutations for carbapenem resistance. Consequently, further studies on this topic are essential. Furthermore, several mutations in the porins OprD, CarO or OmpA were found in the strains analysed in this study. More studies on the influence of porins in A. baumannii on the resistance to antimicrobial compounds are also urgently needed, especially since many porins have not yet been described in this species. Download figure Open in new tab Figure 1 Growth experiment of A. baumannii ATCC 17978 and its mutants with PBP2 mutation. Measurements were performed in 50 ml LB medium at 37°C for 24 h, all cultures were inoculated to an OD 600 of 0.05. The mean value and the standard deviation from three growth experiments are shown. Measurements were made using CGQ. Download figure Open in new tab Figure 2 Overview on PBPs of Acinetobacter baumannii Figure adapted from Lange et al ., 2019 (TP – transpeptidase, GT – glycosylase, CP – carboxypeptidase, EP – endopeptidase Funding This work was supported by the Robert Koch-Institute with funds provided by the German Ministry of Health (grant no. 1369-402). Transparency declarations S.G.G. has received speaker fees from Beckman Coulter and bioMérieux. N.P. has received speaker or consultancy fees from bioMérieux, Pfizer and Shionogi. All other authors: none to declare. Acknowledgements The authors thank Anke Albrecht, Nadine Frey, Susanne Friedrich, Brigitte Hemmerle, Svenja Hirle, Anja Kaminski, Kirsten Krengel, Ulrike Maduch, Lea Rogge, Marion Schmidt, Laura Suppa, and Joanna Waniczek for excellent technical assistance. Footnotes (The study was performed at this institution) References 1. ↵ Almasaudi SB . Acinetobacter spp . as nosocomial pathogens: Epidemiology and resistance features . 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