Dimercaptosuccinic acid in combination with carbapenems against isogenic strains of Escherichia coli producing or not producing a metallo-ß-lactamase in vitro and in murine peritonitis.

BACKGROUND: Carbapenemase-producing Enterobacterales represent a major therapeutic challenge. MBLs, requiring zinc at their catalytic site, could be inhibited by meso-dimercaptosuccinic acid (DMSA), a heavy metal chelator already widely used for treating lead intoxication. OBJECTIVES: To evaluate the activity of carbapenems alone or combined with DMSA against MBL-producing Escherichia coli in a severe murine peritonitis model. METHODS: Isogenic strains of wild-type E. coli CFT073 producing the MBLs NDM-1, VIM-2 and IMP-1, and the control serine carbapenemases OXA-48 and KPC-3 were constructed. MIC determinations and time-kill assays were performed for imipenem, meropenem and ertapenem alone or in combination with DMSA. Infected mice were treated intraperitoneally for 24 h with imipenem, DMSA or their combination. Bacterial counts in peritoneal fluid and spleen were assessed at 24 h. RESULTS: DMSA in combination with each carbapenem caused a significant decrease in the MICs for all MBL-producing strains, in a concentration-dependent manner, but did not provide benefit against non-MBL strains. In mice infected with the NDM-1-producing strain, the combination of imipenem and DMSA significantly reduced bacterial counts in peritoneal fluid (P = 0.0006) and spleen (P < 0.0001), as compared with imipenem alone, with no benefit against the KPC-3-producing and CFT073 strains. DMSA concentrations in plasma of mice were comparable to those obtained in humans with a standard oral dose. CONCLUSIONS: DMSA restores the activity of carbapenems against MBL-producing strains, and its combination with carbapenems appears to be a promising strategy for the treatment of NDM-producing E. coli infections.

Molecular characterization of multidrug-resistance in Gram-negative bacteria from the Peshawar teaching hospital, Pakistan.

Extended-spectrum ß-lactamases, carbapenemases, 16S rRNA methylases conferring pan-drug aminoglycoside resistance and colistin resistance were investigated among Gram-negative bacteria recovered from clinical samples (infections) from 200 individuals hospitalized at the Khyber Teaching Hospital of Peshawar, north Pakistan, from December 2017 to March 2018. Out of 65 isolates recovered, 19% were carbapenem resistant and 16% carried a bla NDM-1 gene, confirming the widespread distribution of NDM producers in this country. The association of the NDM carbapenem-resistance determinant, together with the extended-spectrum ß-lactamase CTX-M-15 and 16S rRNA methylases, was frequent, explaining the multidrug-resistance pattern observed. All isolates remained susceptible to colistin.

Comparison of methods for detection of plasmid-mediated and chromosomally encoded colistin resistance in Enterobacteriaceae.

OBJECTIVES: Because of the emergence of plasmid-mediated (mcr-1 and mcr-2 genes) and chromosomally encoded colistin resistance, reliable methods for detecting colistin resistance/susceptibility in routine laboratories are required. We evaluated the respective performances of the BD Phoenix automated system, the newly developed Rapid Polymyxin NP test and the broth microdilution (BMD) reference method to detect colistin resistance in Enterobacteriaceae, and particularly those producing mcr-1 and mcr-2. METHODS: Colistin susceptibility of 123 enterobacterial clinical isolates (40 colistin-susceptible and 83 colistin-resistant isolates) was tested with the BD Phoenix automated system, the Rapid Polymyxin NP test and the BMD method. Molecular mechanisms responsible for plasmid-mediated and chromosomally encoded colistin resistance mechanisms were investigated by PCR and sequencing. RESULTS: Considering BMD as a reference method, the BD Phoenix system failed to detect ten colistin-resistant isolates (one Escherichia coli, one Klebsiella pneumoniae, seven Enterobacter species and one Salmonella enterica). The Rapid Polymyxin NP test failed to detect the same single E. coli isolate. Those two latter methods detected the 16 E. coli, K. pneumoniae and S. enterica isolates producing the plasmid-encoded mcr-1 and mcr-2. CONCLUSIONS: The BD Phoenix system and the Rapid Polymyxin NP test are reliable techniques for detecting plasmid-mediated mcr-1 and mcr-2-related colistin resistance. However, a high rate of false susceptibility was observed with the BD Phoenix system, indicating that susceptibility results obtained with that system should be confirmed by BMD method. By contrast, the Rapid Polymyxin NP test showed a good agreement with the BMD method, and results were obtained rapidly (within 2 hours). The BMD method should be performed if minimum inhibitory concentration values are needed.

Antimicrobial activity of octenidine against multidrug-resistant Gram-negative pathogens.

Multidrug-resistant (MR) Gram-negative (GN) pathogens pose a major and growing threat for healthcare systems, as therapy of infections is often limited due to the lack of available systemic antibiotics. Well-tolerated antiseptics, such as octenidine dihydrochloride (OCT), may be a very useful tool in infection control to reduce the dissemination of MRGN. This study aimed to investigate the bactericidal activity of OCT against international epidemic clones of MRGN. A set of five different species (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) was studied to prove OCT efficacy without organic load, under "clean conditions" (0.3 g/L albumin) and under "dirty conditions" (3 g/L albumin + 3 mL/L defibrinated sheep blood), according to an official test norm (EN13727). We used five clonally unrelated isolates per species, including a susceptible wild-type strain, and four MRGN isolates, corresponding to either the 3MRGN or 4MRGN definition of multidrug resistance. A contact time of 1 min was fully effective for all isolates by using different OCT concentrations (0.01% and 0.05%), with a bacterial reduction factor of >5 log10 systematically observed. Growth kinetics were determined with two different wild-type strains (A. baumannii and K. pneumoniae), proving a time-dependent efficacy of OCT. These results highlight that OCT may be extremely useful to eradicate emerging highly resistant Gram-negative pathogens associated with nosocomial infections.

Activity of the novel siderophore cephalosporin cefiderocol against multidrug-resistant Gram-negative pathogens.

The novel siderophore cephalosporin cefiderocol (S-649266) with potent activity against Gram-negative pathogens was recently developed (Shionogi & Co., Ltd.). Here, we evaluated the activity of this new molecule and comparators against a collection of previously characterized Gram-negative isolates using broth microdilution panels. A total of 753 clinical multidrug-resistant Gram-negative isolates collected from hospitals worldwide were tested against cefiderocol and antibiotic comparators (ceftolozane-tazobactam [CT], meropenem [MEM], ceftazidime [CAZ], ceftazidime-avibactam [CZA], colistin [CST], aztreonam [ATM], amikacin [AMK], ciprofloxacin [CIP], cefepime [FEP], and tigecycline [TGC]) for their susceptibility. The collection included Escherichia coli (n = 164), Klebsiella pneumoniae (n = 298), Enterobacter sp. (n = 159), Pseudomonas aeruginosa (n = 45), and Acinetobacter baumannii (n = 87). Resistance mechanisms included producers of carbapenemases and extended-spectrum ß-lactamases (ESBLs). In addition, a series of colistin-resistant enterobacterial isolates (n = 74), including 15 MCR-1 producers, were tested. The MIC90 of cefiderocol was 2 mg/L, while those of comparative drugs were >64 mg/L for CT, MEM, CAZ, CZA, and AMK, >32 mg/L for ATM, >16 mg/L for FEP, 8 mg/L for CST, and 2 mg/L for TGC. The MIC50 of cefiderocol was 0.5 mg/L, while those of other drugs were >64 mg/L for CAZ, 64 mg/L for CT, >32 mg/L for ATM, >16 mg/L for FEP, 8 mg/L for MEM and AMK, >4 mg/L for CIP, 1 mg/L for CZA, 0.5 mg/L for TGC, and <0.5 mg/L for CST. Only 20 out of 753 strains showed MIC values of cefiderocol ≥8 µg/mL. Compared to the other drugs tested, cefiderocol was more active, with the exception of colistin and tigecycline showing equivalent activity against certain subgroups of bacteria.

Cross-resistance to human cationic antimicrobial peptides and to polymyxins mediated by the plasmid-encoded MCR-1?

OBJECTIVES: To evaluate whether acquired resistance to cationic antimicrobial peptide (CAMP) group molecules, being normal components of the human immune system, may select co-resistance to antibiotic peptides such as polymyxins, considering they share the same mechanism of action. We aimed to evaluate strains producing the recently identified plasmid-encoded polymyxin resistance determinant MCR-1, which is a phosphoethanolamine transferase that modifies the lipopolysaccharide structure of Gram-negative bacteria. METHODS: In vitro susceptibility studies were performed using human CAMPs, namely cathelicidin LL-37, α-defensin 5 (HD5), and ß-defensin 3 (HDB3), towards MCR-1-producing and colistin-resistant Escherichia coli or Klebsiella pneumoniae. RESULTS: Cross-resistance to CAMPs and colistin mediated by MCR-1 or chromosomal mechanisms was neither observed in E. coli nor in K. pneumoniae. CONCLUSION: Future therapeutic development of human CAMPs is not likely to be impeded by the spread of MCR-1 plasmid-mediated resistance to polymyxins, at least in E. coli.

Extended-spectrum ß-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans?

There has been a great and long-term concern that extended-spectrum ß-lactamase (ESBL)/AmpC- and carbapenemase-producing Enterobacteriaceae occurring in animals may constitute a public-health issue. A large number of factors with complex interrelations contribute to the spread of those bacteria among animals and humans. ESBL/AmpC- or carbapenemase-encoding genes are most often located on mobile genetic elements favouring their dissemination. Some shared reservoirs of ESBL/AmpC or carbapenemase genes, plasmids or clones have been identified and suggest cross-transmissions. Even though exposure to animals is regarded as a risk factor, evidence for a direct transfer of ESBL/AmpC-producing bacteria from animals to humans through close contacts is limited. Nonetheless, the size of the commensal ESBL/AmpC reservoir in non-human sources is dramatically rising. This may constitute an indirect risk to public health by increasing the gene pool from which pathogenic bacteria can pick up ESBL/AmpC/carbapenemase genes. The extent to which food contributes to potential transmission of ESBL/AmpC producers to humans is also not well established. Overall, events leading to the occurrence of ESBL/AmpC- and carbapenemase-encoding genes in animals seem very much multifactorial. The impact of animal reservoirs on human health still remains debatable and unclear; nonetheless, there are some examples of direct links that have been identified.

MCR: modern colistin resistance.

Recently, plasmid-mediated and, therefore, transferable bacterial polymyxin resistance was discovered in strains from both humans and animals. Such a trait may widely spread geographically, while simultaneously crossing microbial species barriers. This may ultimately render the "last resort" polymyxin antibiotics therapeutically useless. Colistin is currently used to treat infections caused by Gram-negative carbapenemase producers and colistin resistance may lead to practical pan-antibiotic resistance. We here analyzed the medical and diagnostic consequences of (emerging) colistin resistance and propose pathways toward adequate diagnostics for timely detection of both asymptomatic carriage and infection. Culture-based testing using chromogenic and selective media for screening clinical (and veterinary) specimens may constitute key tools for that purpose. Relevant molecular tests are also discussed.

First detection of Klebsiella variicola producing OXA-181 carbapenemase in fresh vegetable imported from Asia to Switzerland.

BACKGROUND: The emergence and worldwide spread of carbapenemase-producing Enterobacteriaceae is of great concern to public health services. The aim of this study was to investigate the occurrence of carbapenemase-producing Enterobacteriaceae in fresh vegetables and spices imported from Asia to Switzerland. FINDINGS: Twenty-two different fresh vegetable samples were purchased in March 2015 from different retail shops specializing in Asian food. The vegetables included basil leaves, bergamont leaves, coriander, curry leaves, eggplant and okra (marrow). Samples had been imported from Thailand, the Socialist Republic of Vietnam and India. After an initial enrichment-step, carbapenemase-producing Enterobacteriaceae were isolated from two carbapenem-containing selective media (SUPERCARBA II and Brilliance CRE Agar). Isolates were screened by PCR for the presence of bla KPC, bla NDM, bla OXA-48-like and bla VIM. An OXA-181-producing Klebsiella variicola was isolated in a coriander sample with origin Thailand/Vietnam. The bla OXA-181 gene was encoded in a 14'027 bp region flanked by two IS26-like elements on a 51-kb IncX3-type plasmid. CONCLUSIONS: The results of this study suggest that the international production and trade of fresh vegetables constitute a possible route for the spread of carbapenemase-producing Enterobacteriaceae. The presence of carbapenemase-producing organisms in the food supply is alarming and an important food safety issue.

KPC-producing Klebsiella pneumoniae, finally targeting Turkey.

We report here the first identification of the worldwide spread of Klebsiella pneumoniae carbapenemase-2-producing and carbapenem-resistant K. pneumoniae clone ST258 in Turkey, a country where the distantly-related carbapenemase OXA-48 is known to be endemic. Worryingly, this isolate was also resistant to colistin, now considered to be the last-resort antibiotic for carbapenem-resistant isolates.

The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide.

The spread of carbapenemase producers in Enterobacteriaceae has now been identified worldwide. Three main carbapenemases have been reported; they belong to three classes of ß-lactamases, which are KPC, NDM, and OXA-48. The main reservoirs of KPC are Klebsiella pneumoniae in the USA, Israel, Greece, and Italy, those of NDM are K. pneumoniae and Escherichia coli in the Indian subcontinent, and those of OXA-48 are K. pneumoniae and Escherichia coli in North Africa and Turkey. KPC producers have been mostly identified among nosocomial isolates, whereas NDM and OXA-48 producers are both nosocomial and community-acquired pathogens. Control of their spread is still possible in hospital settings, and relies on the use of rapid diagnostic techniques and the strict implemention of hygiene measures.

[Emerging and important antibiotic resistance in Gram negative bacteria: epidemiology, theory and practice]. / Résistances aux antibiotiques émergentes et importantes chez les bactéries Gram négatif: épidémiologie, aspects théoriques et détection.

Emerging and clinically-relevant antibiotic resistance mechanisms among Gram-negative rods are the extended-spectrum beta-lactamases (ESBL), carbapenemases, and 16S RNA methylases conferring resistance to aminoglycosides. Those resistance determinants do confer multiresistance to antibiotics. They are found in Enterobacteriaceae (especially community-acquired isolates, Pseudomonas aeruginosa and Acinetobacter baumannii). Detection of ESBL-producing and carbapenemase-producing isolates rely on the use of rapid diagnostic techniques that have to be performed when a reduced susceptibility to 3rd/4th generation cephalosporins or to carbapenems is observed, respectively. Only an early detection of those emerging resistance traits may contribute to limit their nosocomial spread and to optimize the antibiotic stewardship.

Rapid detection of carbapenemase-producing Enterobacteriaceae from blood cultures.

The biochemical-based Carba NP test has been evaluated to detect carbapenemase-producing Enterobacteriaceae (n = 193) directly from spiked blood cultures. It was able to rapidly detect KPC (n = 50), IMP (n = 27), VIM (n = 37), NDM (n = 33) and OXA-48-like producers (n = 46) with sensitivity and specificity of 97.9% and 100%, respectively. This cost-effective technique may be implemented in any microbiology laboratory and offers a reliable test for an early identification of carbapenemase-producing Enterobacteriaceae directly from blood culture that could be useful for the management of infected patients.

High rate of faecal carriage of extended-spectrum ß-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae at a university hospital in Morocco.

Carbapenemase-producing Enterobacteriaceae isolates are being increasingly reported, particularly from countries surrounding the Mediterranean area. We aimed to quantify the prevalence of carbapenemase- and extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in rectal swabs from hospitalized patients in a University hospital in Morocco, and to compare the performance of three screening media: ChromID ESBL (bioMérieux), Brilliance CRE (OXOID, Thermofisher) and SUPERCARBA (home made). Genetic detection and plasmid analysis were performed by PCR and sequencing. Strain comparison was performed by multi-locus sequence typing and the Diversilab technique (bioMérieux). The prevalence of multidrug-resistant Enterobacteriaceae was high, with 33 ESBL producers (42.85%, mainly CTX-M-15) and 10 OXA-48 producers (13%), corresponding to two major clones of K. pneumoniae (70%) and a clone of Enterobacter cloacae (30%). The three screening media showed the same sensitivity for detection of carbapenemase-producing Enterobacteriaceae, whereas the SUPERCARBA medium was more specific than the two other media. The average faecal carriage of ESBL or carbapenemase-producing Enterobacteriaceae varied from 1 × 10(2) to >1 × 10(8) CFU/g of stools. This study shows a high prevalence of multidrug-resistant Enterobacteriaceae, and particularly of OXA-48 producers. The new carbapenem-containing medium, SUPERCARBA, was as sensitive as Brilliance CRE and ChromID ESBL, and more specific for the detection of Enterobacteriaceae expressing those carbapenemases.

Outbreak of NDM-1-producing Acinetobacter baumannii in France, January to May 2013.

We report the first outbreak of carbapenem-resistant NDM-1-producing Acinetobacter baumannii in Europe, in a French intensive-care unit in January to May 2013. The index patient was transferred from Algeria and led to the infection/colonisation of five additional patients. Concurrently, another imported case from Algeria was identified. The seven isolates were genetically indistinguishable, belonging to ST85. The bla(NDM-1) carbapenemase gene was part of the chromosomally located composite transposon Tn125. This report underscores the growing concern about the spread of NDM-1-producing A. baumannii in Europe.

Intercontinental spread of OXA-48 beta-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011.

OXA-48 beta-lactamase producers are emerging as an important threat mostly in the Mediterranean area. We report here the molecular epidemiology of a collection of OXA-48 beta-lactamase-positive enterobacterial isolates (n=107) recovered from European and north-African countries between January 2001 and December 2011. This collection included 67 Klebsiella pneumoniae, 24 Escherichia coli and 10 Enterobacter cloacae. Using the EUCAST breakpoints, ninety-eight isolates (91.6%) were of intermediate susceptibility or resistant to ertapenem, whereas 66% remained susceptible to imipenem. Seventy-five per cent of the isolates co-produced an extended-spectrum beta-lactamase, most frequently CTX-M-15 (77.5%). Susceptibility testing to non-beta-lactam antibiotics showed that colistin, tigecycline, amikacin, and fosfomycin remain active against most of the isolates. Multilocus sequence typing indicated that the most common sequence types (ST) were ST101 and ST38 for K. pneumoniae and E. coli, respectively. The bla(OXA-48) gene was located on a 62 kb IncL/M plasmid in 92.5% of the isolates, indicating that a single plasmid was mainly responsible for the spread of that gene. In addition, this study identified multiple cases of importation of OXA-48 beta-lactamase producers at least in Europe, and spread of OXA-48 beta-lactamase producers giving rise to an endemic situation, at least in France.