[Methicillin-resistant Staphylococcus aureus: 15 years of molecular epidemiology in Western Switzerland]. / Staphylococcus aureus résistant à la méticilline : 15 ans de surveillance moléculaire en Suisse romande.

Since the introduction of antibiotics, successive waves of Staphylococcus aureus clones occurred, each one having characteristic susceptibility pattern to antibiotics and virulence factors. We report here the results of a molecular epidemiological surveillance of methicillin-resistant S. aureus (MRSA) in French-speaking Switzerland between 2006 and 2020 showing the emergence and disappearance of clones known for their international dissemination, and the sporadic appearance of other international clones. Since 2012, a marked decrease in the incidence of cases attributable to the biology of the clones and to the control measures taken in the hospitals has been observed. These results highlight the importance of continuous surveillance in order to better assess the burden of this multi-resistant pathogen in our region.

Depuis l'introduction des antibiotiques, des vagues successives de clones de Staphylococcus aureus sont apparues, chacun avec un profil de susceptibilité aux antibiotiques et de virulence caractéristique. Nous rapportons ici les résultats d'une surveillance épidémiologique moléculaire de S. aureus résistant à la méticilline (MRSA) en Suisse romande entre 2006 et 2020 montrant l'émergence et la disparition de clones connus pour leur dissémination internationale, ainsi que l'apparition sporadique d'autres clones internationaux. Depuis 2012, une diminution marquée de l'incidence des cas attribuable à la biologie des clones et aux mesures de contrôle prises dans les hôpitaux est observée. Ces résultats nous montrent l'importance d'une surveillance continue afin de mieux évaluer le fardeau que représente ce germe multirésistant dans notre région.

Efficacy of colistin alone and in various combinations for the treatment of experimental osteomyelitis due to carbapenemase-producing Klebsiella pneumoniae.

OBJECTIVES: In a new experimental model of carbapenemase-producing Klebsiella pneumoniae osteomyelitis we evaluated the efficacy of colistin alone and in various combinations and examined the emergence of colistin-resistant strains and cross-resistance to host defence peptides (HDPs). METHODS: KPC-99YC is a clinical strain with intermediate susceptibility to meropenem (MIC = 4 mg/L) and full susceptibility to gentamicin, colistin and tigecycline (MICs = 1 mg/L) and fosfomycin (MIC = 32 mg/L). Time-kill curves were performed at 4× MIC. Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu. Treatment started 14 days later for 7 days in seven groups: (i) control; (ii) colistin; (iii) colistin + gentamicin; (iv) colistin + tigecycline; (v) colistin + meropenem; (vi) colistin + meropenem + gentamicin; and (vii) colistin + fosfomycin. RESULTS: In vitro, colistin was rapidly bactericidal, but regrowth occurred after 9 h. Combinations of colistin with meropenem or fosfomycin were synergistic, whereas combination with tigecycline was antagonistic. In vivo, colistin alone was not effective. Combinations of colistin with meropenem or fosfomycin were bactericidal (P < 0.001) and the addition of gentamicin enhanced the efficacy of colistin + meropenem (P = 0.025). Tigecycline reduced the efficacy of colistin (P = 0.007). Colistin-resistant strains emerged in all groups except colistin + fosfomycin and two strains showed cross-resistance to HDP LL-37. CONCLUSIONS: In this model, combinations of colistin plus meropenem, with or without gentamicin, or colistin plus fosfomycin were the only effective therapies. The combination of colistin and tigecycline should be administered with caution, as it may be antagonistic in vitro and in vivo.

Performances of the Rapid Polymyxin Acinetobacter and Pseudomonas Tests for Colistin Susceptibility Testing.

Objectives: Owing to the emergence of colistin resistance in nonfermenting Gram negative bacteria, reliable and rapid techniques for testing colistin susceptibility are needed. We evaluated the performances of the Rapid Polymyxin Acinetobacter and Pseudomonas tests using a collection of Acinetobacter baumannii and Pseudomonas aeruginosa clinical isolates. Methods: Colistin susceptibility of A. baumannii and P. aeruginosa isolates (colistin susceptible and colistin resistant) was tested with the Rapid Polymyxin Acinetobacter and Pseudomonas tests and compared with the broth microdilution method. Results: The Rapid Polymyxin Acinetobacter and Pseudomonas tests were able to detect all colistin-resistant and all colistin-susceptible A. baumannii and P. aeruginosa isolates within 4 hours. Conclusion: The Rapid Polymyxin Acinetobacter and Pseudomonas tests are reliable techniques for detecting colistin resistance. Overall, both techniques allow an accurate and a rapid screening (<4 hours) of colistin resistance in A. baumannii and P. aeruginosa.

Acquisition of Extended-Spectrum ß-Lactamase GES-6 Leading to Resistance to Ceftolozane-Tazobactam Combination in Pseudomonas aeruginosa.

A clinical Pseudomonas aeruginosa isolate resistant to all ß-lactams, including ceftolozane-tazobactam and carbapenems, was recovered. It belonged to sequence type 235 and produced the extended-spectrum ß-lactamase (ESBL) GES-6 differing from GES-1 by two amino acid substitutions (E104K and G170S). GES-6 possessed an increased hydrolytic activity toward carbapenems and to ceftolozane and a decreased susceptibility to ß-lactamase inhibitors compared to GES-1, except for avibactam. We show here that resistance to ceftolozane-tazobactam may occur through acquisition of a specific ESBL in P. aeruginosa but that ceftazidime-avibactam combination remains an effective alternative.

Evaluation of the Rapid Polymyxin NP test and its industrial version for the detection of polymyxin-resistant Enterobacteriaceae.

The commercial Rapid Polymyxin NP test was evaluated to detect colistin-resistant Enterobacteriaceae. A total of 223 enterobacterial isolates corresponding to 136 resistant (including 38 MCR-like producers), 19 heteroresistant, and 78 colistin-susceptible isolates were tested. The test was performed according to the manufacturer's instruction, and the color of the wells was read after 2 and 3 hours of incubation. The results were compared with those of the homemade Rapid Polymyxin NP test, and manual broth microdilution according to EUCAST guidelines was used as the reference method to determine the performance of the test. Excellent performance of the commercial Rapid Polymyxin NP test was found with a very major error rate, a major error rate, a sensitivity, and a specificity of 1.9%, 5.1%, 98.1%, and 94.9%, respectively. The performance of the homemade Polymyxin NP test was similar, with a slightly better value for the very major error (1.2 %).

Detection of colistin-resistant Gram-negative rods by using the SuperPolymyxin medium.

The commercial SuperPolymyxin medium (ELITechGroup, Puteaux, France), currently licensed in Europe, is aimed to screen for either intrinsic or acquired colistin resistance in Gram negatives. It was tested for screening colistin-resistant Gram-negative rods from bacterial cultures of 145 colistin-resistant and 86 colistin-susceptible Gram-negative rods isolates. It exhibited a sensitivity and specificity of 95.2% (138/145) and 95.3% (82/86), respectively. The sensitivity for the detection of the 25 isolates carrying plasmid-mediated colistin resistance genes (mcr-1, mcr-2, mcr-3, and mcr-4) from bacterial cultures was of 100%. It was also evaluated for the detection of colistin-resistant Gram-negative rods from 57 rectal swab samples (41 clinical rectal swab samples and from 16 spiked rectal swab samples supplemented with colistin-resistant enterobacterial isolates carrying mcr genes). The overall sensitivity and specificity were of 100% (33/33) and 90.3% (56/62), respectively. The detection of isolates carrying plasmid-mediated mcr-like genes from rectal swabs was achieved with a lowest detection limit of 103 to 104 CFU/mL. The SuperPolymyxin medium is adapted for screening and detection of colistin-resistant isolates from bacterial cultures and rectal swab samples, regardless of the level and the mechanism of colistin resistance. It might be useful in the context of prevention of outbreaks due to colistin-resistant isolates and for epidemiological surveillance.

A culture medium for screening 16S rRNA methylase-producing pan-aminoglycoside resistant Gram-negative bacteria.

The amikacin plus gentamicin-containing SuperAminoglycoside medium was developed for screening multiple-aminoglycoside resistance in Gram-negative bacteria (Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii). It was evaluated using aminoglycoside-susceptible (n=12) and aminoglycoside-resistant (n=59) Gram-negative isolates, including 16S rRNA methylase producers (n=20). Its sensitivity and specificity of detection were, respectively, of 95% and 96% for detecting multiple aminoglycoside-resistant methylase producers.

Are animals a source of Stenotrophomonas maltophilia in human infections? Contributions of a nationwide molecular study.

Stenotrophomonas maltophilia (Sm) is an archetypal environmental opportunistic bacterium responsible for health care-associated infections. The role of animals in human Sm infections is unknown. This study aims to reveal the genetic and phylogenetic relationships between pathogenic strains of Sm, both animal and human, and identify a putative role for animals as a reservoir in human infection. We phenotypically and genotypically characterized 61 Sm strains responsible for animal infections (mainly respiratory tract infections in horses) from a French nationwide veterinary laboratory network. We tested antimicrobial susceptibility and performed MLST and genogrouping using the concatenation of the seven housekeeping genes from the original MLST scheme. Excluding the eight untypeable strains owing to the lack of gene amplification, only 10 out of the 53 strains yielded a known ST (ST5, ST39, ST162, ST8, ST27, ST126, ST131). The genogroup distribution highlighted not only genogroups (genogroups 5 and 9) comprised exclusively of animal strains but also genogroups shared by human and animal strains. Interestingly, these shared genogroups were primarily groups 2 and 6, which have previously been identified as the two most frequent genogroups among human-pathogenic Sm strains, especially among respiratory pathogens. The antimicrobial susceptibility testing underlined the presence of acquired resistance: 18.8 and 7.5% of the tested isolates were resistant to the sulfonamide-trimethoprim combination and ciprofloxacin, respectively. Animal strains of Sm shared phylogenetic traits with some of the most successful human strains. The exact relationships between the human and animal strains, and the genetic support of these common traits, need to be determined.

Evaluation of three broth microdilution systems to determine colistin susceptibility of Gram-negative bacilli.

Background: The broth microdilution (BMD) method is currently the recommended technique to determine susceptibility to colistin. Objectives: We evaluated the accuracy of three commercialized BMD panels [Sensititre (ThermoFisher Diagnostics), UMIC (Biocentric) and MicroScan (Beckman Coulter)] to determine colistin susceptibility. Methods: A collection of 185 isolates of Gram-negative bacilli (133 colistin resistant and 52 colistin susceptible) was tested. Manual BMD according to EUCAST guidelines was used as the reference method, and EUCAST 2017 breakpoints were used for susceptibility categorization. Results: The UMIC system gave the highest rate of very major errors (11.3%) compared with the Sensititre and MicroScan systems (3% and 0.8%, respectively). A high rate of major errors (26.9%) was found with the MicroScan system due to an overestimation of the MICs for the non-fermenting Gram-negative bacilli, whereas no major errors were found with the Sensititre and UMIC systems. Conclusions: The UMIC system was easy to use, but failed to detect >10% of colistin-resistant isolates. The MicroScan system showed excellent results for enterobacterial isolates, but non-susceptible results for non-fermenters should be confirmed by another method and the range of MICs tested was narrow. The Sensititre system was the most reliable marketed BMD panel with a categorical agreement of 97.8%.

High-Level Resistance to Colistin Mediated by Various Mutations in the crrB Gene among Carbapenemase-Producing Klebsiella pneumoniae.

Mutations in crrAB genes encoding a two-component regulator involved in modifications of lipopolysaccharide were searched for among a collection of colistin-resistant Klebsiella pneumoniae isolates. Four isolates, respectively, producing carbapenemases NDM-1, OXA-181, or KPC-2 showed mutated CrrB proteins compared with those in wild-type strains. Complementation assays with a wild-type CrrB protein restored the susceptibility to colistin in all cases, confirming the involvement of the identified substitutions in the resistance phenotype.

Hafnia, an enterobacterial genus naturally resistant to colistin revealed by three susceptibility testing methods.

Objectives: To determine the susceptibility to colistin of Hafnia alvei and Hafnia paralvei, and to compare methods for colistin resistance detection in the Hafnia genus. Methods: A collection of 25 Hafnia isolates was studied. Species were identified by using 16S rRNA gene sequencing with subsequent phylogeny analysis. Susceptibility to colistin was determined using the broth microdilution (BMD) reference method, the Phoenix automated system, the Rapid Polymyxin NP test, the Etest system and the disc diffusion method. Results: The collection consisted of 15 H. alvei and 10 H. paralvei isolates. Based on the 16S rRNA analysis, a close relationship of the Hafnia genus with naturally colistin-resistant enterobacterial genera (Proteus, Morganella, Providencia and Serratia) was identified. Susceptibility testing performed using the BMD method, the Phoenix automated system and the Rapid Polymyxin NP test revealed a high rate of colistin resistance (96%). Underestimation of colistin resistance using Etest strips (72%) and the disc diffusion method (0%) was observed. Conclusions: The high rate of colistin resistance observed within the Hafnia genus and its close phylogenetic relationship with naturally colistin-resistant genera suggest that Hafnia is a naturally colistin-resistant enterobacterial genus.

Prevalence of faecal carriage of colistin-resistant Gram-negative rods in a university hospital in western France, 2016.

Plasmid-mediated and chromosomally-encoded colistin resistance is increasingly being reported worldwide. We aimed to determine the prevalence of faecal carriage of colistin-resistant Gram-negative rod isolates in a university hospital in western France. From February to May 2016, rectal swabs from 653 patients hospitalized in various clinical settings were recovered and subsequently screened for colistin resistance using the SuperPolymyxin medium. Antimicrobial susceptibilities were determined according to EUCAST guidelines. Genetic detection of plasmid-mediated colistin resistance was performed by PCR. The faecal carriage with intrinsic colistin-resistant isolates was high (23 %), while the faecal carriage with Gram-negative rods showing acquired resistance was low (1.4 %). No isolate carried the plasmid-mediated mcr-1/mcr-2 genes. It was noteworthy that none of the patients carrying isolates with acquired colistin resistance had previously received a colistin-based treatment, while these isolates were not multidrug resistant.

Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes.

Polymyxins are well-established antibiotics that have recently regained significant interest as a consequence of the increasing incidence of infections due to multidrug-resistant Gram-negative bacteria. Colistin and polymyxin B are being seriously reconsidered as last-resort antibiotics in many areas where multidrug resistance is observed in clinical medicine. In parallel, the heavy use of polymyxins in veterinary medicine is currently being reconsidered due to increased reports of polymyxin-resistant bacteria. Susceptibility testing is challenging with polymyxins, and currently available techniques are presented here. Genotypic and phenotypic methods that provide relevant information for diagnostic laboratories are presented. This review also presents recent works in relation to recently identified mechanisms of polymyxin resistance, including chromosomally encoded resistance traits as well as the recently identified plasmid-encoded polymyxin resistance determinant MCR-1. Epidemiological features summarizing the current knowledge in that field are presented.

Rapid Aminoglycoside NP Test for Rapid Detection of Multiple Aminoglycoside Resistance in Enterobacteriaceae.

The rapid aminoglycoside NP (Nordmann/Poirel) test was developed to rapidly identify multiple aminoglycoside (AG) resistance in Enterobacteriaceae It is based on the detection of the glucose metabolism related to enterobacterial growth in the presence of a defined concentration of amikacin plus gentamicin. Formation of acid metabolites was evidenced by a color change (orange to yellow) of the red phenol pH indicator. The rapid aminoglycoside NP test was evaluated by using bacterial colonies of 18 AG-resistant isolates producing 16S rRNA methylases, 20 AG-resistant isolates expressing AG-modifying enzymes (acetyl-, adenyl-, and phosphotransferases), and 10 isolates susceptible to AG. Its sensitivity and specificity were 100% and 97%, respectively, compared to the broth dilution method, which was taken as the gold standard for determining aminoglycoside resistance. The test is inexpensive, rapid (<2 h), and implementable worldwide.

National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014.

From January 2014 to December 2014, 972 consecutive non-replicate carbapenemase-producing Enterobacteriaceae isolates from colonised or infected patients were collected at the Associated French National Reference Centre as part of the French national survey on antimicrobial resistance. It included 577 Klebsiella spp. (59%), 236 Escherichia coli (24%), 108 Enterobacter spp. (11%), 50 Citrobacter spp. (5%), and a single Salmonella spp. isolate (0.1%). Of 561 K. pneumoniae isolates, 35 were found to be resistant to colistin (6.2%). PFGE analysis revealed a clonal outbreak involving 15 K. pneumoniae isolates belonging to sequence type ST11, recovered in a single hospital in the Picardie region in northern France. Those clonally related isolates showed variable levels of resistance to colistin, ranging from 4 to 64 mg/L. They harboured the blaOXA-48 carbapenemase gene and the blaCTX-M-15 extended-spectrum beta-lactamase gene. Among the 91 Enterobacter cloacae isolates, seven were resistant to colistin and produced different types of carbapenemases. Surprisingly, none of the E. coli and Citrobacter spp. isolates showed resistance to colistin. This national survey including carbapenemase-producing isolates recovered in 2014 reported a high rate of colistin resistance in K. pneumoniae and E. cloacae (6.2% and 7.7%, respectively) in France.

Rapid Detection of Polymyxin-Resistant Enterobacteriaceae from Blood Cultures.

Enterobacterial strains resistant to polymyxins are being increasingly reported worldwide. The conventional methods for detection of colistin-resistant isolates such as broth microdilution remain time-consuming (24 to 48 h), and methods such as disc diffusion and Etest are not reliable. Recently, the rapid polymyxin NP test was developed for rapid identification of polymyxin-resistant Enterobacteriaceae This test is based on the detection of glucose metabolism related to bacterial growth in the presence of a defined concentration of colistin (or polymyxin B). The formation of acid metabolites is evidenced by a color change of a pH indicator (red phenol) in less than 2 h. In this study, the polymyxin NP test was evaluated for detection of colistin-resistant Enterobacteriaceae directly from blood cultures. The test was performed with 73 blood culture sets (either spiked or clinical blood cultures) with various enterobacterial species. The test exhibited excellent discrimination between polymyxin-resistant and polymyxin-susceptible enterobacterial isolates, and results are obtained from blood cultures within 4 h. It is easy to perform and requires neither subculture nor a centrifugation step. This test is rapid, specific, and sensitive and allows early identification of polymyxin-resistant Enterobacteriaceae directly from blood cultures.

Rapid Detection of Polymyxin Resistance in Enterobacteriaceae.

For identification of polymyxin resistance in Enterobacteriaceae, we developed a rapid test that detects glucose metabolization associated with bacterial growth in the presence of a defined concentration of colistin or polymyxin B. Formation of acid metabolites is evidenced by a color change (orange to yellow) of a pH indicator (red phenol). To evaluate the test, we used bacterial colonies of 135 isolates expressing various mechanisms of colistin resistance (intrinsic, chromosomally encoded, and plasmid-mediated MCR-1) and 65 colistin-susceptible isolates. Sensitivity and specificity were 99.3% and 95.4%, respectively, compared with the standard broth microdilution method. This new test is inexpensive, easy to perform, sensitive, specific, and can be completed in <2 hours. It could be useful in countries facing endemic spread of carbapenemase producers and for which polymyxins are last-resort drugs.

Genetic Features of MCR-1-Producing Colistin-Resistant Escherichia coli Isolates in South Africa.

A series of colistin-resistant Escherichia coli clinical isolates was recovered from hospitalized and community patients in South Africa. Seven clonally unrelated isolates harbored the mcr-1 gene located on different plasmid backbones. Two distinct plasmids were fully sequenced, and identical 2,600-bp-long DNA sequences defining a mcr-1 cassette were identified. Promoter sequences responsible for the expression of mcr-1, deduced from the precise identification of the +1 transcription start site for mcr-1, were characterized.