Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes

Colistin resistance has attracted substantial attention after colistin was considered as a last-resort drug for the treatment of infections caused by carbapenem-resistant and/or multidrug-resistant (MDR) Gram-negative bacteria in clinical settings. However, with the discovery of highly mobile colistin resistance (mcr) genes, colistin resistance has become an increasingly urgent issue worldwide. Despite many reviews, which summarized the prevalence, mechanisms, and structures of these genes in bacteria of human and animal origin, studies on the prevalence of mobile colistin resistance genes in aquaculture and their transmission between animals and humans remain scarce. Herein, we review recent reports on the prevalence of colistin resistance genes in animals, especially wildlife and aquaculture, and their possibility of transmission to humans via the food chain. This review also gives some insights into the routine surveillance, changing policy and replacement of polymyxins by polymyxin derivatives, molecular inhibitors, and traditional Chinese medicine to tackle colistin resistance.     

Global distribution of plasmid-mediated colistin resistance mcr gene in Salmonella: A systematic review

This systematic review focuses on obtaining the most relevant information from multiple studies that detected a mobilized colistin resistance mcr gene in Salmonella for a better comprehension of its global distribution. A group of strategic and systematic keywords were combined to retrieve research data on the detection frequency of the mcr gene globally from four database platforms (Google Scholar, Science Direct, PubMed and Scielo). Forty-eight studies attended all the eligibility criteria and were selected. China was the country with the highest frequency of Salmonella strains with the mcr gene, and Europe exhibited a wide diversity of countries with positive mcr strains. In addition, animals and humans carried the highest frequency of positive strains for the mcr gene. Salmonella Typhimurium was the most frequent serovar carrying the mcr gene. Apparently, colistin overuse in animal husbandry has increased the selective pressure of antimicrobial resistance, resulting in the emergence of a plasmid-mediated colistin resistance mcr gene in China. The mcr-positive Salmonella strains are recently predominant worldwide, which is probably due to the capacity of this gene to be swiftly horizontally transmissible. The transmission ability of mcr-positive Salmonella strains to humans through the consumption of contaminated animal-based food is a public health concern.     

Comparison of disc diffusion, Etest and agar dilution for susceptibility testing of colistin against Enterobacteriaceae

Aims: In this study, we compared different methods of colistin susceptibility testing, disc diffusion, agar dilution and Etest using a set of Enterobacteriaceae isolates that included colistin‐resistant strains. Methods and results: Susceptibility of 200 clinical isolates of Enterobacteriaceae to colistin was tested to compare agar dilution (reference method), disc diffusion (50 and 10 μg) and Etest. MICs (minimum inhibitory concentrations) were interpreted using the criteria established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin exhibited excellent activity against Escherichia coli and E. cloacae (MIC₉₀ = 0·5 mg l^?1). In contrast, colistin was less active against Klebsiella pneumoniae (MIC₉₀ = 16 mg l^?1). Resistance rates varied from 0% in E. coli to 1·8% in E. cloacae and 13% in K. pneumoniae. High rates of very major errors were observed in the disc diffusion test using either the criteria of the Comité de l’antibiogramme de la Société Française de Microbiologie (CA‐SFM) or the criteria of the Clinical and Laboratory Standards Institute (CLSI), respectively, 3·5 and 2·5%. When the criteria of Gales et al. were applied, the number of very major errors was reduced to one (0·5%). The Etest showed good concordance with agar dilution method. Conclusion: Disc susceptibility testing methods are unreliable on detecting colistin resistance. MIC should be determined to confirm the susceptibility results by disc diffusion. Significance and Impact of the study: We recommend the determination of MIC by Etest for all multidrug‐resistant Enterobacteriaceae when colistin is required for the treatment.     

Enzymatic Degradation of Colistin Isolation and Identification of α-N-Acyl α,γ-Diaminobutyric Acid and Colistin Nonapeptide

Colistin, a fatty acyl peptide antibiotic, was attacked by proteolytic enzymes such as papain, ficin and bromelain, and as degradation product, a peptide portion retaining the ring structure of colistin was liberated. In contrast, an analogous antibiotic polymyxin B showed a characteristic resistance to the catalytic activity of papain.

Colistin nonapeptide and α-N-fatty acyl α,γ-diaminobutyric acid were obtained as products from the above enzymatic hydrolyzates of colistin and their chemical and physicochemical properties were investigated.

Contrary to colistin, this colistin nonapeptide was inactive to Escherichia coli. NIHJ and to many other strains even at a concentration of 800 mcg/ml by the agar dilution method. As α-N-fatty acyl α,γ-diaminobutyric acid which is rest part of colistin was added to colistin nonapeptide, antimicrobial activity of colistin nonapeptide did not increase.     

Performance of different methods for testing polymyxin B: comparison of broth microdilution,agar dilution and MIC test strip in mcr-1 positive and negative Escherichia coli

Antimicrobial susceptibility testing with the last-resort antibiotics polymyxins (polymyxin B and colistin) is associated with several methodological issues. Currently, broth microdilution (BMD) is recommended for colistin and polymyxin B. BMD is laborious and the utility of alternative methods needs to be evaluated for polymyxin B susceptibility testing. In this study, using BMD as a reference method, the performance of agar dilution (AD) and MIC test strips (MTS) were evaluated in polymyxin B susceptibility testing. BMD, AD and MTS were used to determine MICs of 193 clinical isolates of Escherichia coli. Seventy-nine were positive for the polymyxin resistance gene mcr-1. Method performances were evaluated based on pair-wise agreements with the reference method (BMD) and statistical testing. AD and MTS showed an unacceptable number of very major errors (VMEs) compared with BMD, 9·3 and 10·7%, respectively. The essential agreement (EA) was low for AD (49·7%), but high for MTS (97·8%). However, statistical testing showed that MTS tended to yield a one-step lower MIC (P < 0·01) compared with BMD. The discordances observed with MTS and AD in comparison with BMD for polymyxin B susceptibility testing for E. coli suggest their inapplicability in routine testing. A large number of isolates clustered around the susceptibility breakpoint (2–4 mg l⁻¹) and several mcr-1 positive isolates (17%) were determined as susceptible with BMD. A screening breakpoint for mcr-1 of 2 mg l⁻¹ should also be considered.     

Induced colistin resistance as an identifying marker for Aeromonas phenospecies groups

AIMS: To investigate the taxonomic interest of colistin resistance as an identifying marker for Aeromonas phenospecies groups. METHODS AND RESULTS: Colistin resistance was investigated in 387 Aeromonas isolates identified at species level using a 14-test format protocol with miniaturized tests combined with determination of urocanic acid utilization whenever necessary. Colistin resistance, determined by the disc diffusion method, was unreliable when compared with minimum inhibitory concentration (MIC) determination. In some strains, the MIC values and resistance rates of colistin could be increased after overnight induction with a 50- microg colistin disc in 20 ml of Mueller-Hinton broth (2.5 mg l(-1)). Colistin-induced resistance level was raised to 85.8% in the Aeromonas hydrophila complex, 2.1% in the A. caviae complex and 2.5% in the A. veronii complex except for A. jandaei (100% colistin resistant). This new marker allowed the identification of 96.2 and 93.6% of Aeromonas isolates to phenospecies and species level, respectively. CONCLUSIONS: Colistin-induced colistin resistance is a new phenotypic marker for Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: With the present protocol, colistin resistance determination may improve the identification of Aeromonas isolates to phenogroup level, when results obtained by conventional biochemical methods are ambiguous.     

Performance of polymyxin B agar-based tests among carbapenem-resistant Enterobacterales

Therapeutic options for infections caused by Carbapenem-resistant Enterobacterales (CRE) are restricted and include polymyxins-centred schemes. Evaluation of in vitro susceptibility is difficult and time consuming. Agar-based methodologies are an alternative to broth microdilution (BMD) and we aimed to evaluate the accuracy of those methods among Enterobacterales. A total of 137 non-duplicated CRE were subjected to polymyxin B BMD, agar screening test (Mueller Hinton plates containing 3 µg ml⁻¹ of polymyxin B) and agar dilution (antibiotic serially diluted 0·25–64 µg ml⁻¹). CRE of 42·3% were resistant to polymyxin B (MICs range: 0·25–>64 µg ml⁻¹) and 16·8% presented borderline MICs. Sensitivity, specificity, PPV and NPV were 86·2, 98·7, 98 and 90·7% for screening test and 86·2, 97·5, 96·1 and 90·6% for agar dilution. ME was 0·73 and 1·5% for screening and agar dilution respectively; VME was 5·8% for both techniques. In general, agar-based methods had a good performance. As far as we know, this is the first study to propose an agar screening test using polymyxin B instead of colistin.     

Association of the colistin resistance gene mcr-1 with faecal pollution in water environments in Hanoi,Vietnam

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.     

Effects of Colistin and Bacteriocins Combinations on the In Vitro Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains from Swine Origin

Escherichia coli strains from swine origin, either susceptible or resistant to colistin, were grown under planktonic and biofilm cultures. After which, they were treated with antibacterial agents including nisin and enterocin DD14 bacteriocins, colistin and their combinations. Importantly, the combination of colistin, enterocin DD14 and nisin eradicated the planktonic and biofilm cultures of E. coli CIP54127 and the E. coli strains with colistin-resistance phenotype such as E. coli 184 (mcr-1 ⁺) and E. coli 289 (mcr-1 ^?), suggesting therefore that bacteriocins from lactic acid bacteria could be used as agents with antibiotic augmentation capability.     

Screening of mcr-1 Among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria,Egypt: One-Year Study

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2^® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla_OXA-48, and three of them co-harbored bla_NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat. Open in a separate window     

Extended-spectrum beta-lactamase-producing Escherichia coli harbouring sul and mcr-1 genes isolates from fish gut contents in the Mekong Delta,Vietnam

This study investigated the existence of sulfonamides and colistin resistance genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli recovered from fish gut in Vietnam and evaluated the susceptibility patterns of the ESBL-producing E. coli to relevant antimicrobials. A total of 88 ESBL-producing E. coli isolates were analysed for the presence of the ESBLs, sul (1, 2, 3) and mcr (1–3) genes by PCR. Antimicrobial resistance phenotypes of isolates were determined by disc diffusion. Results showed that: (i) A high prevalence of 94·3% of sulfonamide resistance was observed in 88 isolates. Moreover, the existence of 2·3% of ESBL-producing E. coli harbouring mcr-1 gene were detected; (ii) The phylogenetic types A and B1 were most frequent, and the bla_CTX-M group1 and bla_TEM genes encoding ESBL were detected in 47·7% of the isolates; (iii) ESBL-producing E. coli harbouring mcr-1 gene exhibited resistance to 11 antibiotics. The existence of mcr-1 and sul1,2,3 genes and the extremely high level of multiple drug resistance in all ESBL-producing E. coli isolates obtained from sampled fish in Vietnam is a major concern. Therefore, it is imperative to monitor ESBL-producing E. coli in the river waters of Vietnam.     

Co-production of Tet(X) and MCR-1, two resistance enzymes by a single plasmid

Tigecycline and colistin are few of ‘last-resort’ antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.     

A rapid MALDI-TOF mass spectrometry-based method for colistin susceptibility testing in Escherichia coli

Colistin is recognized as a last-resort treatment option against multi-drug resistant bacteria including carbapenem-resistant Enterobacteriaceae (CRE). However, the plasmid-mediated colistin-resistance gene mcr-1 has been reported globally resulting in an increase of colistin-resistant bacteria. A quick and accurate method for determining the pathogen resistance of colistin is therefore crucial in the clinic. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a potential tool forto be applied for antimicrobial susceptibility testing. We compared the growth of Escherichia coli strains in the presence or absence of colistin. Automated analyses of the spectra were performed with a prototype software tool written with package R. Three mcr-1-positive and six mcr-1-negative E. coli were used for establishing the model to obtain the optimal incubation time, the breakpoint concentration of colistin and cut-off of the relative growth (RG) value. The distinction between susceptible and resistant strains was already noticeable after 2 h of incubation. The best separation between the susceptible and resistant strains was achieved at a concentration of 4 µg ml^-1 and a relative growth cut-off value of 0.6. Application of the model for the analysis of 128 E. coli isolates, a sensitivity of 97.4% and a specificity of 88.2% were achieved compared with colistin MIC results. The rapid MALDI-TOF MS-based method approach is simple to set-up, uses a short incubation time, and had excellent outcomes with respect to sensitivity and specificity for colistin sensitivity testing in Escherichia coli.     

Cell surface hydrophobicity of colistin-susceptible vs resistant Acinetobacter baumannii determined by contact angles: methodological considerations and implications

Contact angle analysis of cell surface hydrophobicity (CSH) describes the tendency of a water droplet to spread across a lawn of filtered bacterial cells. Colistin‐induced disruption of the Gram‐negative outer membrane necessitates hydrophobic contacts with lipopolysaccharide (LPS). We aimed to characterize the CSH of Acinetobacter baumannii using contact angles, to provide insight into the mechanism of colistin resistance. Contact angles were analysed for five paired colistin‐susceptible and resistant Ac. baumannii strains. Drainage of the water droplet through bacterial layers was demonstrated to influence results. Consequently, measurements were performed 0·66 s after droplet deposition. Colistin‐resistant cells exhibited lower contact angles (38·8±2·8–46·8±1·3°) compared with their paired colistin‐susceptible strains (40·7±3·0–48·0±1·4°; anova ; P < 0·05). Contact angles increased at stationary phase (50·3±2·9–61·5±2·5° and 47·4±2·0–50·8±3·2°, susceptible and resistant, respectively, anova ; P < 0·05) and in response to colistin 32 mg l^?1 exposure (44·5±1·5–50·6±2·8° and 43·5±2·2–48·0±2·2°, susceptible and resistant, respectively; anova ; P < 0·05). Analysis of complemented strains constructed with an intact lpxA gene, or empty vector, highlighted the contribution of LPS to CSH. Compositional outer‐membrane variations likely account for CSH differences between Ac. baumannii phenotypes, which influence the hydrophobic colistin–bacterium interaction. Important insight into the mechanism of colistin resistance has been provided. Greater consideration of contact angle methodology is necessary to ensure accurate analyses are performed.     

粘杆菌素发酵液微滤膜分离处理过程研究

粘杆菌素由于其药性强、残留低、对人畜无害被认为是最安全的畜禽抗生素之一。利用微滤对粘杆菌素发酵液进行预处理,根据发酵液的特性,选择孔径为0．2μm、膜面积为0．06M。的管式陶瓷膜为微滤膜,研究了操作参数的适宜值：压力为0．2MPa,流量为20L／min,在浓缩倍数达到3．5倍时连续两次加入与浓缩液体等量的水,得率为96％。经微滤处理后,滤液的吸光度Abs（470nm）为0．394,N-NH,含量为115mg／100mL,有效地去除了菌体、胶体蛋白及部分色素等,效果优于工业生产中板框过滤滤液的质量标准。     

The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi‐resistant clinical strains. In 2‐D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin‐resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.     

Co-occurrence of genes encoding carbapenemase,ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried bla_NDM-1 (n = 11), bla_OXA-48 (n = 11), bla_NDM-1 + bla_OXA-48 (n = 1), bla_NDM-1 + bla_VIM-1 (n = 1), bla_OXA-204 (n = 1), along with bla_CTX-M, bla_OXA, bla_TEM, bla_CMY, bla_DHA and bla_SHV genes variants on conjugative plasmid of IncL/M, IncR, IncFII_K, IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.     

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B₃. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B₃ for LPS was comparable to polymyxin B and colistin, and considerably greater (K_d < 1 μM) than for whole cells (K_d ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B₃ to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B₃–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B₃–LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.     

Studies on the Bacterial Formation of a Peptide Antibiotic,Colistin

Colistinase, an enzyme inactivating colistin, which appeared in a fermented broth of Bacillus colistinus attending with decay of colistin production was studied. The enzyme was partially purified and also chromatographed by DEAE-cellulose column. It was a heat labile, mostly basic protein, had optimum pH at 9.0 and was active against peptide antibiotics such as colistin and polymyxin and also against casein. Bacterial proteinase, Nagarse, exhibited colistinase activity exclusively among tested proteinases and immunochemical studies revealed that colistinase was identical with bacterial proteinase Nagarse.