[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.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

Acquisition of Broad-Spectrum Cephalosporin Resistance Leading to Colistin Resistance in Klebsiella pneumoniae.

An extended-spectrum ß-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a blaCTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad-spectrum cephalosporin selective pressure.

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.

A Universal Culture Medium for Screening Polymyxin-Resistant Gram-Negative Isolates.

The colistin-containing SuperPolymyxin medium was developed for screening polymyxin-resistant Gram-negative bacteria. It was evaluated with 88 polymyxin-susceptible or polymyxin-resistant cultured Gram-negative isolates. Its sensitivity and specificity of detection were ca. 100%. The SuperPolymyxin medium is the first screening medium that is able to detect intrinsic and acquired polymyxin-resistant bacteria.

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.

Chromobacterium spp. harbour Ambler class A ß-lactamases showing high identity with KPC.

OBJECTIVES: The origin of KPC is unknown. The aim of this study was to detect progenitors of KPC in silico and to functionally verify their ß-lactam hydrolysis activity. METHODS: The sequence of KPC-2 was used to mine the NCBI protein sequence database. The best non-KPC hits were analysed by amino acid (aa) alignment and phylogenetic tree construction. Genes encoding KPC-2 homologues were expressed in Escherichia coli. The carbapenemase activities of the recombinant strains were characterized by the CarbaNP test and UV spectrophotometry and MICs of selected ß-lactams were determined. RESULTS: Genes encoding the closest KPC-2 homologues were identified on the chromosome of Chromobacterium piscinae strain ND17 (CRP-1, 76% aa identity), Chromobacterium sp. C-61 (CRS-1, 70% aa identity) and Chromobacterium haemolyticum DSM19808 (CRH-1, 69% aa identity). All three Chromobacterium ß-lactamases were phylogenetically more related to KPC than to other Ambler class A ß-lactamases. The 27 bp region preceding the start codon of blaCRP-1 displayed high nucleotide identity to the corresponding region upstream from blaKPC (74%). Heterologous expression of blaCRP-1 and to a lesser extent of blaCRH-1 in E. coli significantly increased the MICs of meropenem and most cephalosporins. The CarbaNP test was positive for both recombinant strains, but spectrophotometric analysis confirmed higher carbapenemase activity for CRP-1-producing clones. CONCLUSIONS: The recovery of three class A ß-lactamases with up to 76% aa identity to KPC from distinct Chromobacterium species is highly indicative of the role played by this genus in the evolution of KPC.

qnrA6 genetic environment and quinolone resistance conferred on Proteus mirabilis.

OBJECTIVES: To determine the genetic location and environment of the qnrA6 gene in Proteus mirabilis PS16 where it was first described and to characterize the quinolone resistance qnrA6 confers. METHODS: Transformation experiments and Southern blotting were performed for plasmid and genomic DNA of P. mirabilis PS16 to determine the qnrA6 location. Combinatorial PCRs with primers in qnrA6 and genes usually surrounding qnrA genes were used to determine the genetic environment. The qnrA6 coding region, including or not the promoter region, was cloned into vectors pTOPO and pBR322 and the MICs of six quinolones were measured for transformants of Escherichia coli TOP10 and P. mirabilis ATCC 29906 Rif(R). RESULTS: qnrA6 was shown to be chromosomally encoded in P. mirabilis PS16 and its genetic environment was 81%-87% similar to that of qnrA2 in the Shewanella algae chromosome. The 5138 bp region up- and downstream of qnrA6 contained an IS10 sequence surrounded by two ISCR1. This resulted in qnrA6 being displaced 1.9 kb from its native promoter but supplied a promoter present in ISCR1. qnrA6 cloned into pTOPO and pBR322 conferred a 4-32-fold increase in fluoroquinolone MICs when expressed in E. coli but only 2-3-fold in P. mirabilis. When including the promoter region, a further increase in resistance was observed in both species, reaching MIC values above clinical breakpoints for only P. mirabilis. CONCLUSIONS: qnrA6 is the first chromosomally located qnrA gene described in Enterobacteriaceae. The quinolone resistance conferred by qnrA6 depends on the proximity of an efficient promoter and the host strain where it is expressed.

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.

Heteroresistance to colistin in Klebsiella pneumoniae associated with alterations in the PhoPQ regulatory system.

A multidrug-resistant Klebsiella pneumoniae isolate exhibiting heteroresistance to colistin was investigated. The colistin-resistant subpopulation harbored a single amino acid change (Asp191Tyr) in protein PhoP, which is part of the PhoPQ two-component system that activates pmrHFIJKLM expression responsible for l-aminoarabinose synthesis and polymyxin resistance. Complementation assays with a wild-type phoP gene restored full susceptibility to colistin. Then, analysis of the colistin-susceptible subpopulation showed a partial deletion (25 bp) in the phoP gene compared to that in the colistin-resistant subpopulation. That deletion disrupted the reading frame of phoP, leading to a longer and inactive protein (255 versus 223 amino acids long). This is the first report showing the involvement of mutation(s) in PhoP in colistin resistance. Furthermore, this is the first study to decipher the mechanisms leading to colistin heteroresistance in K. pneumoniae.

The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae.

OBJECTIVES: Alterations in the PhoPQ two-component regulatory system may be associated with colistin resistance in Klebsiella pneumoniae. MgrB is a small transmembrane protein produced upon activation of the PhoPQ signalling system, and acts as a negative regulator on this system. We investigated the role of the MgrB protein as a source of colistin resistance in a series of K. pneumoniae. METHODS: Colistin-resistant K. pneumoniae isolates were recovered from hospitalized patients worldwide (France, Turkey, Colombia and South Africa). The mgrB gene was amplified and sequenced. A wild-type mgrB gene was cloned and the corresponding recombinant plasmid was used for complementation assays. Clonal diversity was evaluated by MLST and Diversilab analysis. RESULTS: Of 47 colistin-resistant isolates, 12 were identified as having a mutated mgrB gene. Five clonally unrelated isolates had an mgrB gene truncated by an IS5-like IS, while one clone also harboured an insertional inactivation at the exact same position of the mgrB gene, but with ISKpn13. Another clone harboured an insertional inactivation due to ISKpn14 at another location of the mgrB gene. Two clonally related isolates harboured an IS (IS10R) in the promoter region of mgrB. Finally, three clonally unrelated isolates harboured substitutions leading to anticipated stop codon in the MgrB protein. Complementation assays with a wild-type MgrB protein restored full susceptibility to colistin for all colistin-resistant isolates identified with qualitative or quantitative MgrB modifications. CONCLUSION: The inactivation or down-regulation of the mgrB gene was shown to be a source of colistin resistance in K. pneumoniae. Interestingly, identical genetic events were identified among clonally unrelated isolates.

Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin.

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.