Impact of clonal lineages on susceptibility of Staphylococcus lugdunensis to chlorhexidine digluconate and chloride benzalkonium.

BACKGROUND: Little is known about susceptibility of Staphylococcus lugdunensis to antiseptics. The objective of this study was to evaluate, at the molecular and phenotypic level, the susceptibility of 49 clinical S. lugdunensis strains (belonging to the seven clonal complexes [CCs] defined by multilocus sequence typing) to two antiseptics frequently used in healthcare settings (chlorhexidine digluconate [CHX] and chloride benzalkonium [BAC]). RESULTS: The minimum inhibitory concentrations (MICs), by broth microdilution method, varied for BAC from 0.25 mg/L to 8 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L) and for CHX from 0.5 mg/L to 2 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L). The BAC and CHX minimum bactericidal concentrations (MBCs) varied from 2 mg/L to 8 mg/L (MBC50 = 4 mg/L, MBC90 = 8 mg/L) and from 2 mg/L to 4 mg/L (MBC50 and MBC90 = 4 mg/L), respectively. A reduced susceptibility to CHX (MIC = 2 mg/L) was observed for 12.2% of the strains and that to BAC (MIC ≥ 4 mg/L) for 4.1%. The norA resistance gene was detected in all the 49 isolates, whereas the qacA gene was rarely encountered (two strains; 4.1%). The qacC, qacG, qacH, and qacJ genes were not detected. The two strains harboring the qacA gene had reduced susceptibility to both antiseptics and belonged to CC3. CONCLUSION: The norA gene was detected in all the strains, suggesting that it could belong to the core genome of S. lugdunensis. S. lugdunensis is highly susceptible to both antiseptics tested. Reduced susceptibility to BAC and CHX was a rare phenomenon. Of note, a tendency to higher MICs of BAC was detected for CC3 isolates. These results should be confirmed on a larger collection of strains.

Are Escherichia coli causing recurrent cystitis just ordinary Uropathogenic E. coli (UPEC) strains?

Specific determinants associated with Uropathogenic Escherichia coli (UPEC) causing recurrent cystitis are still poorly characterized. The aims of this study were (i) to describe genomic and phenotypic traits associated with recurrence using a large collection of recurrent and paired sporadic UPEC isolates, and (ii) to explore within-host genomic adaptation associated with recurrence using series of 2 to 5 sequential UPEC isolates. Whole genome comparative analyses between 24 recurrent cystitis isolates (RCIs) and 24 phylogenetically paired sporadic cystitis isolates (SCIs) suggested a lower prevalence of putative mobile genetic elements (MGE) in RCIs, such as plasmids and prophages. The intra-patient evolution of the 24 RCI series over time was characterized by SNP occurrence in genes involved in metabolism or membrane transport, and by plasmid loss in 5 out of the 24 RCI series. Genomic evolution occurred early in the course of recurrence, suggesting rapid adaptation to strong selection pressure in the urinary tract. However, RCIs did not exhibit specific virulence factor determinants and could not be distinguished from SCIs by their fitness, biofilm formation, or ability to invade HTB-9 bladder epithelial cells. Taken together, these results suggest a rapid but not convergent adaptation of RCIs that involves both strain- and host-specific characteristics.

Contribution of Antibiotic Susceptibility Testing and CH Typing Compared to Next-Generation Sequencing for the Diagnosis of Recurrent Urinary Tract Infections Due to Genetically Identical Escherichia coli Isolates: a Prospective Cohort Study of Cystitis in Women.

Recurrent cystitis is a common disease in women, mainly due to uropathogenic Escherichia coli (UPEC). For decades, typing methods now considered obsolete suggested that relapse by the same clone is dominant over reinfection, most UPEC strains being otherwise fully susceptible to antibiotics. We aimed to update these data. Thanks to a prospective study over 17 months, we recruited 323 women with cystitis. Of these, 251 of them had sporadic infection and 72 had recurrence, with 2 to 9 episodes per patient for a total of 131 UPEC isolates and 145 UPEC pairs at patient level. Phylogroups B2 (52.4%) and D (14.1%) were overall dominant, as expected due to their particular urovirulence. CH typing identified 119 distinct profiles with no CH type particularly associated with recurrence. Relapse was attested by CH typing for only 30.6% (22 out of 72), with very diverse situations ranging from all episodes due to the same clone to alternating reinfections and relapses. Next-generation sequencing confirmed the clonality for all but two of the 145 UPEC pairs. Antibiotic resistance was common for recurrent cystitis isolates (only 25 [17.2%] out of 145 UPEC pairs were fully susceptible), allowing us to predict UPEC clonality. Indeed, antibiotic susceptibility profile matched CH typing for 104 (71.7%) pairs. Finally, we demonstrated a large genetic diversity among UPEC isolates responsible for cystitis in women, even in cases of recurrence for which reinfection appeared dominant over relapse. Recurrent cystitis appears to be a heterogeneous disease requiring tailored treatment and prevention. IMPORTANCE More than half of women will experience cystitis during their lifetime. Among these women, 25% will experience a second episode within the following 6 months. It is epidemiologically important to discriminate relapses from reinfections. Relapse identification relies on long and laborious methods and might influence treatment. Therefore, the designation of time- and cost-effective strategies for this goal is of particular interest. Our work suggests using CH typing and antibiotic susceptibility profiles to type Escherichia coli, the main uropathogen.

Performance evaluation of UF-4000 body fluid mode for detection of bacteria in body fluids.

BACKGROUND: Microbiological analysis of body fluids (BF) provides important information for diagnosis of infection. We evaluated the analytical performance of bacterial count by UF-4000 BF mode for ascitic, cerebrospinal, pleural, synovial and continuous ambulatory peritoneal dialysis fluids compared to classical microbiological procedure (direct Gram staining and culture). MATERIALS AND METHODS: For the 1,734 BF analyzed, distribution of UF-4000 bacterial count was analyzed according to the level of growth culture and results were compared using Mann-Whitney test. ROC curves analysis allowed to define the best cut-off value to predict or exclude positive culture for each type of BF. RESULTS: UF-4000 bacterial counts were significantly lower in sterile than in infected BFs (p < 0.00001) and correlated with the level of growth on culture. The ROC curves of bacteria/µL and culture positivity yielded area under the curve >0.80 for each type of BF. Optimal cut-offs were chosen with excellent statistical parameters (sensitivity ranging from 0.70 to 0.86, specificity from 0.78 to 0.98, negative predictive value >0.95 and Youden index >0.55). CONCLUSION: For BF, UF-4000 bacterial count correlate with culture results and is a discriminative method enhancing detection of microbiological etiology. It could be used as a screening method based on the cut-off values proposed in this study.

Performance evaluation of UF-4000 body fluid mode for automated body fluid cell counting.

BACKGROUND: Cytological analysis of body fluids (BF) provides important information for diagnosis in various medical conditions. We evaluated the analytical performance of the UF-4000 BF mode for ascitic, cerebrospinal, pleural, synovial and continuous ambulatory peritoneal dialysis fluids compared to light microscopy counting (LM). MATERIALS AND METHODS: 223 consecutive BF were analyzed by UF-4000 and results were compared using Pearson's correlation, Bland-Altman analysis, and contingence tests at relevant cut-off values. This study also included the evaluation of precision, linearity, and carryover. RESULTS: For white and red blood cells (WBC, RBC) counts in all BF, correlation was excellent with Pearson's coefficients R2 > 0,98. Bland-Altman analysis didn't reveal significant differences with limited bias for WBC ranging from -10 to -1 WBC/µL and bias ranging from -43 to -6/µL for RBC. At specific cut-off values for WBC, Se and Spe were 100% except for ascites (Spe = 98%) due to two false positive. Precision evaluated at three concentration levels was good for each parameter (WBC < 10%). Linearity was excellent for WBC (R2 > 0,99) and carryover negligible (<0,004%). CONCLUSION: UF-4000 BF mode is a good alternative to manual LM for BF cell counting. This automated method gives rapid and accurate results which is important for therapeutic decisions.

Phenotypic and genotypic within-host diversity of Pseudomonas aeruginosa urinary isolates.

This study aimed to assess phenotypic and molecular inter-patient and within-host diversity of Pseudomonas aeruginosa isolates responsible for urinary tract infection (UTI) or asymptomatic bacteriuria (AB). Clinical data of 120 consecutive P. aeruginosa UTI (n = 40) and AB (n = 80) were prospectively analyzed. Up to five P. aeruginosa isolates per sample were collected. Antimicrobial susceptibility testing (AST) was determined for all isolates (n = 591); a subset of 358 was characterized by multilocus sequence typing. 444 isolates (75%) were non-multidrug resistant (MDR), 113 (19%) were MDR, and 34 (6%) were extensively drug resistant. A genetically highly diverse population was observed (64 sequence types [STs]), without strict correlation between genotypes and clinical settings. 35 patients (28%; 12 UTIs and 23 ABs) presented distinct antimicrobial resistance (AMR) profiles within a given urine sample, significantly associated with previous carbapenem and fluroquinolones exposure; five of them also exhibited polyclonal UTI or AB (with isolates belonging to two STs). P. aeruginosa urinary isolates of these 120 patients were highly diverse, in terms of AMR as well as genetic background. Both within-host AMR and molecular diversity can complicate AST, treatment and control of P. aeruginosa UTI.

Efficacy of temocillin against MDR Enterobacterales: a retrospective cohort study.

OBJECTIVES: EUCAST recently advised against temocillin use, except for non-serious urinary tract infections (UTI) caused by Escherichia coli, Klebsiella spp. (except Klebsiella aerogenes) and Proteus mirabilis (EKP) treated with a dose of 2 g q8h. We aimed to analyse our practice in the context of a larger temocillin use in France. PATIENTS AND METHODS: All ≥3 day temocillin prescriptions from 2016 to 2019 were reviewed, with reference to French recommendations and a susceptibility breakpoint of 8 mg/L. The primary outcome was early clinical failure (antibiotic switch, relapse or death within 10 days after the completion of antibiotic treatment). RESULTS: Overall, 153 cases were analysed: 123 cases of UTI (80.4%) and 133 cases of monomicrobial infection with Enterobacterales (86.9%). A total of 160 Enterobacterales were isolated, comprising 108 (67.5%) ESBL producers and 30 (20.7%) non-EKP species. The rate of early clinical failure was 9.2% and was significantly lower for UTI compared with non-UTI (4.9% versus 26.7%, P = 0.001) and for sepsis compared with severe sepsis or septic shock (6.2% versus 25%, P = 0.011). It was not different between 2 g q12h and 2 g q8h doses (10% versus 7.4%, P = 0.81) and between EKP and other Enterobacterales (8.7% versus 14.3%, P = 0.41). CONCLUSIONS: EUCAST recommendations on urinary isolates seem to be too restrictive. Our data support the efficacy of temocillin at a dose of 2 g q12h to treat patients with non-severe complicated UTI caused by MDR Enterobacterales with an MIC of ≤8 mg/L, whatever the species.

Role of the LytSR Two-Component Regulatory System in Staphylococcus lugdunensis Biofilm Formation and Pathogenesis.

Staphylococcus lugdunensis is a coagulase negative Staphylococcus recognized as a virulent pathogen. It is responsible for a wide variety of infections, some of which are associated with biofilm production, such as implanted medical device infections or endocarditis. However, little is known about S. lugdunensis regulation of virulence factor expression. Two-component regulatory systems (TCS) play a critical role in bacterial adaptation, survival, and virulence. Among them, LytSR is widely conserved but has variable roles in different organisms, all connected to metabolism or cell death and lysis occurring during biofilm development. Therefore, we investigated here the functions of LytSR in S. lugdunensis pathogenesis. Deletion of lytSR in S. lugdunensis DSM 4804 strain did not alter either susceptibility to Triton X-100 induced autolysis or death induced by antibiotics targeting cell wall synthesis. Interestingly, ΔlytSR biofilm was characterized by a lower biomass, a lack of tower structures, and a higher rate of dead cells compared to the wild-type strain. Virulence toward Caenorhabditis elegans using a slow-killing assay was significantly reduced for the mutant compared to the wild-type strain. By contrast, the deletion of lytSR had no effect on the cytotoxicity of S. lugdunensis toward the human keratinocyte cell line HaCaT. Transcriptional analyses conducted at mid- and late-exponential phases showed that lytSR deletion affected the expression of 286 genes. Most of them were involved in basic functions such as the metabolism of amino acids, carbohydrates, and nucleotides. Furthermore, LytSR appeared to be involved in the regulation of genes encoding known or putative virulence and colonization factors, including the fibrinogen-binding protein Fbl, the major autolysin AtlL, and the type VII secretion system. Overall, our data suggest that the LytSR TCS is implicated in S. lugdunensis pathogenesis, through its involvement in biofilm formation and potentially by the control of genes encoding putative virulence factors.

Within-Host Microevolution of Pseudomonas aeruginosa Urinary Isolates: A Seven-Patient Longitudinal Genomic and Phenotypic Study.

BACKGROUND: Pseudomonas aeruginosa is responsible for up to 10% of healthcare associated urinary tract infections (UTI), which can be difficult to treat and can lead to bacterial persistence. While numerous whole genome sequencing (WGS) analyses have explored within-host genomic adaptation and microevolution of P. aeruginosa during cystic fibrosis (CF) infections, little is known about P. aeruginosa adaptation to the urinary tract. RESULTS: Whole genome sequencing was performed on 108 P. aeruginosa urinary isolates, representing up to five isolates collected from 2 to 5 successive urine samples from seven patients hospitalized in a French hospital over 48-488 days. Clone type single nucleotide polymorphisms (ctSNPs) analysis revealed that each patient was colonized by a single clone type (<6000 SNPs between two isolates) at a given time and over time. However, 0-126 SNPs/genome/year were detected over time. Furthermore, large genomic deletions (1-5% of the genome) were identified in late isolates from three patients. For 2 of them, a convergent deletion of 70 genes was observed. Genomic adaptation (SNPs and deletion) occurred preferentially in genes encoding transcriptional regulators, two-component systems, and carbon compound catabolism. This genomic adaptation was significantly associated with a reduced fitness, particularly in artificial urine medium, but no strict correlation was identified between genomic adaptation and biofilm formation. CONCLUSION: This study provides the first insight into P. aeruginosa within-host evolution in the urinary tract. It was driven by mutational mechanisms and genomic deletions and could lead to phenotypic changes in terms of fitness and biofilm production. Further metabolomic and phenotypic analyses are needed to describe in-depth genotype-phenotype associations in this complex and dynamic host-environment.

Comparative Genome Analysis of Staphylococcus lugdunensis Shows Clonal Complex-Dependent Diversity of the Putative Virulence Factor, ess/Type VII Locus.

Staphylococcus lugdunensis is a commensal bacterium of human skin that has emerged as a virulent Coagulase-Negative Staphylococcus in both community-acquired and healthcare associated infections. Genotyping methods have shown a clonal population structure of this pathogen but failed to identify hypervirulent lineages. Here, complete genomes of three pathogenic and three carriage S. lugdunensis strains were obtained by Single-Molecule sequencing (PacBio) and compared to 15 complete genomes available in GenBank database. The aim was to identify (i) genetic determinants specific to pathogenic or carriage strains or specific to clonal complexes (CCs) defined by MultiLocus Sequence Typing, and (ii) antibiotic resistance genes and new putative virulence factors encoded or not by mobile genetic elements (MGE). Comparative genomic analysis did not show a strict correlation between gene content and the ability of the six strains to cause infections in humans and in a Galleria mellonella infection model. However, this study identified new MGEs (five prophages, two genomic islands and one plasmid) and genetic variations of some putative virulence-associated loci, especially in CC3 strains. For a clonal population, high variability and eight CC-dependent genetic organizations were observed for the ess locus, which encodes a putative type VII secretion system (T7SS) homologous to that of S. aureus. Further phenotypic and functional studies are needed to characterize this particular CC3 and to evaluate the role of T7SS in the virulence of S. lugdunensis.

fbl-Typing of Staphylococcus lugdunensis: A Frontline Tool for Epidemiological Studies, but Not Predictive of Fibrinogen Binding Ability.

Staphylococcus lugdunensis is increasingly recognized as a potent pathogen, responsible for severe infections with an outcome resembling that of Staphylococcus aureus. Here, we developed and evaluated a tool for S. lugdunensis typing, using DNA sequence analysis of the repeat-encoding region (R-domain) in the gene encoding the fibrinogen (Fg)-binding protein Fbl (fbl-typing). We typed 240 S. lugdunensis isolates from various clinical and geographical origins. The length of the R-domain ranged from 9 to 52 repeats. fbl-typing identified 54 unique 18-bp repeat sequences and 92 distinct fbl-types. The discriminatory power of fbl-typing was higher than that of multilocus sequence typing (MLST) and equivalent to that of tandem repeat sequence typing. fbl-types could assign isolates to MLST clonal complexes with excellent predictive power. The ability to promote adherence to immobilized human Fg was evaluated for 55 isolates chosen to reflect the genetic diversity of the fbl gene. We observed no direct correlation between Fg binding ability and fbl-types. However, the lowest percentage of Fg binding was observed for isolates carrying a 5'-end frameshift mutation of the fbl gene and for those harboring fewer than 43 repeats in the R-domain. qRT-PCR assays for some isolates revealed no correlation between fbl gene expression and Fg binding capacity. In conclusion, this study shows that fbl-typing is a useful tool in S. lugdunensis epidemiology, especially because it is an easy, cost-effective, rapid and portable method (http://fbl-typing.univ-rouen.fr/). The impact of fbl polymorphism on the structure of the protein, its expression on the cell surface and in virulence remains to be determined.

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Tandem Repeat Sequence Typing (TRST), helpful tools for subtyping Staphylococcus lugdunensis.

Staphylococcus lugdunensis is an emergent virulent coagulase-negative Staphylococcus that is increasingly responsible for severe infections. In an attempt to generate informative sequence data for subtyping S. lugdunensis, we selected and sequenced seven polymorphic variable number of tandem repeats (VNTRs) to develop two new methods: a classic length-based multiple-locus VNTR analysis (MLVA) method and a tandem repeat sequence typing (TRST) method. We assessed their performances compared to two existing methods, multilocus sequence typing (MLST) and multivirulence-locus sequence typing (MVLST) for 128 isolates from diverse clinical settings and geographical origins. The clustering achieved by the four methods was highly congruent, with MLVA discriminating within clonal complexes as defined by MLST. Indeed, MLVA was highly discriminant compared to MLST and MVLST in terms of number of genotypes as well as diversity indexes. Sequencing of the seven VNTRs showed that they were stable, and analysis of sequence polymorphisms provided superior discriminatory power. The typeability, reproducibility, and epidemiological concordance of these new methods were excellent. Of note, no link between clustering and clinical settings was identified. This study demonstrates that MLVA and TRST provide valuable information for molecular epidemiological study of S. lugdunensis, and represent promising tools to distinguish between strains of homogenous lineages in this clonal species.

Comparative genomic analysis of Staphylococcus lugdunensis shows a closed pan-genome and multiple barriers to horizontal gene transfer.

BACKGROUND: Coagulase negative staphylococci (CoNS) are commensal bacteria on human skin. Staphylococcus lugdunensis is a unique CoNS which produces various virulence factors and may, like S. aureus, cause severe infections, particularly in hospital settings. Unlike other staphylococci, it remains highly susceptible to antimicrobials, and genome-based phylogenetic studies have evidenced a highly conserved genome that distinguishes it from all other staphylococci. RESULTS: We demonstrate that S. lugdunensis possesses a closed pan-genome with a very limited number of new genes, in contrast to other staphylococci that have an open pan-genome. Whole-genome nucleotide and amino acid identity levels are also higher than in other staphylococci. We identified numerous genetic barriers to horizontal gene transfer that might explain this result. The S. lugdunensis genome has multiple operons encoding for restriction-modification, CRISPR/Cas and toxin/antitoxin systems. We also identified a new PIN-like domain-associated protein that might belong to a larger operon, comprising a metalloprotease, that could function as a new toxin/antitoxin or detoxification system. CONCLUSION: We show that S. lugdunensis has a unique genome profile within staphylococci, with a closed pan-genome and several systems to prevent horizontal gene transfer. Its virulence in clinical settings does not rely on its ability to acquire and exchange antibiotic resistance genes or other virulence factors as shown for other staphylococci.

Whole-Genome Sequencing of Seven Strains of Staphylococcus lugdunensis Allows Identification of Mobile Genetic Elements.

Coagulase negative staphylococci are normal inhabitant of the human skin flora that account for an increasing number of infections, particularly hospital-acquired infections. Staphylococcus lugdunensis has emerged as a most virulent species causing various infections with clinical characteristics close to what clinicians usually observe with Staphylococcus aureus and both bacteria share more than 70% of their genome. Virulence of S. aureus relies on a large repertoire of virulence factors, many of which are encoded on mobile genetic elements. S. lugdunensis also bears various putative virulence genes but only one complete genome with extensive analysis has been published with one prophage sequence (φSL2) and a unique plasmid was previously described. In this study, we performed de novo sequencing, whole genome assembly and annotation of seven strains of S. lugdunensis from VISLISI clinical trial. We searched for the presence of virulence genes and mobile genetics elements using bioinformatics tools. We identified four new prophages, named φSL2 to φSL4, belonging to the Siphoviridae class and five plasmids, named pVISLISI_1 to pVISLISI_5. Three plasmids are homologous to known plasmids that include, amongst others, one S. aureus plasmid. The two other plasmids were not described previously. This study provides a new context for the study of S. lugdunensis virulence suggesting the occurrence of several genetic recombination' with other staphylococci.

Contribution of asparagine 346 residue to the carbapenemase activity of CMY-2 ß-lactamase.

Only a few plasmid-borne AmpC (pAmpC) ß-lactamases, such as CMY-2, can account for carbapenem resistance in Enterobacteriaceae in combination with outer membrane impermeability. The aim of this study was to elucidate the contribution of Asn-346, which is well conserved among carbapenem-hydrolyzing pAmpCs, to the hydrolysis spectrum of CMY-2. Site-directed mutagenesis experiments were carried out to replace Asn-346 with glycine, alanine, valine, glutamate, aspartate, serine, threonine, glutamine, tyrosine, isoleucine, lysine, and histidine. The recombinant plasmids were transferred into wild-type and porin-deficient Escherichia coli strains. Asn-346 replacement reduced significantly the MICs of all ß-lactams, except the Asn-346-Ile substitution that increased the MICs of cephalosporins, whereas it decreased those of carbapenems. The biochemical characterization, along with a molecular modeling study, showed that the size and the polarity of the side chain at position 346 assisted substrate binding and turnover. This study shows for the first time that the amino acid at position 346 contributes to the ß-lactamase activity of cephalosporinases. Asparagine and isoleucine residues, which are well conserved at position 346 among AmpC-type enzymes, modulate their hydrolysis spectrum in an opposing sense. Ile-346 confers higher level of cephalosporins resistance, whereas Asn-346 confers carbapenem resistance in combination with outer membrane impermeability.

Hydrolysis spectrum extension of CMY-2-like ß-lactamases resulting from structural alteration in the Y-X-N loop.

The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) ß-lactamase. The chromosome-borne bla(AmpC-CHA) gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 ß-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (k(cat)/K(m)) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (k(cat)), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A ß-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.