Comprehensive Approaches for the Search and Characterization of Staphylococcins.

Novel and sustainable approaches are required to curb the increasing threat of antimicrobial resistance (AMR). Within the last decades, antimicrobial peptides, especially bacteriocins, have received increased attention and are being explored as suitable alternatives to antibiotics. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria as a self-preservation method against competitors. Bacteriocins produced by Staphylococcus, also referred to as staphylococcins, have steadily shown great antimicrobial potential and are currently being considered promising candidates to mitigate the AMR menace. Moreover, several bacteriocin-producing Staphylococcus isolates of different species, especially coagulase-negative staphylococci (CoNS), have been described and are being targeted as a good alternative. This revision aims to help researchers in the search and characterization of staphylococcins, so we provide an up-to-date list of bacteriocin produced by Staphylococcus. Moreover, a universal nucleotide and amino acid-based phylogeny system of the well-characterized staphylococcins is proposed that could be of interest in the classification and search for these promising antimicrobials. Finally, we discuss the state of art of the staphylococcin applications and an overview of the emerging concerns.

Widespread of the Vienna/Hungarian/Brazilian CC8-ST239-SCCmec III MRSA clone in patients hospitalized in the Tunisian Burn and Traumatology Center.

The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals is a major global public health concern. The current study sought to characterize 25 MRSA clinical isolates collected in a Tunisian hospital from December 2015 to September 2016, with the genetic lineages, virulence factors, and antibiotic resistance mechanisms determined for these isolates. Three spa-types were detected: t037 (23 isolates), t932, and t2235 (one isolate each). Isolates were ascribed to agr I (n = 20), agr II (n = 1), with four nontypeable isolates. Depending on sequence type (ST), the 25 MRSA isolates were assigned to two clonal complexes (CC8 and CC5), with a predominance of the lineage ST239-CC8 (n = 24; 96%). All isolates belonging to CC8 had the SCCmec type III, while the unique CC5 isolate had SCCmec type IV. Antimicrobial susceptibility testing revealed high levels of resistance to aminoglycosides, tetracycline, ciprofloxacin and rifampicin for the majority of isolates belonging to the ST239-CC8 lineage. The ST149-CC5 isolate was susceptible to non-ß-lactam antibiotics. One isolate harbored the tsst-1 gene (4%); however, lukS/LukF-PV, eta and etb genes were not detected. The MDR ST239-CC8 clone would seem to be widespread in this hospital. Therefore, a rigorous hygienic control system is urgently required.

Antimicrobial Resistance and Antimicrobial Activity of Staphylococcus lugdunensis Obtained from Two Spanish Hospitals.

Staphylococcus lugdunensis is a coagulase-negative-staphylococci (CoNS) that lately has gained special attention in public health as a human pathogen and also as a bacteriocin-producer bacteria. In this study, we characterized 56 S. lugdunensis isolates recovered from human samples in two Spanish hospitals. Antimicrobial susceptibility testing was performed and antimicrobial resistance and virulence genotypes were determined. Antimicrobial activity (AA) production was evaluated by the spot-on-lawn method against 37 indicator bacteria, including multidrug-resistant (MDR) isolates, and the presence of the lugD gene coding for lugdunin bacteriocin was analyzed by PCR. The antibiotic resistance detected was as follows (% resistance/genes detected): penicillin (44.6%/blaZ), oxacillin (1.8%/mecA on SCCmec-V), erythromycin-clindamycin inducible (7.1%/erm(C), msrA), tetracycline (5.3%/tetK), gentamicin and/or tobramycin (3.6%/ant(4')-Ia, acc(6')-aph(2″)), and fosfomycin (21.4%). A MDR phenotype was detected in 5% of isolates. Twenty-one of the S. lugdunensis isolates showed susceptibility to all 20 antibiotics tested (37.5%). The screening for AA revealed 23 antimicrobial producer (AP) isolates with relevant inhibition against coagulase-positive-staphylococci (CoPS), including both methicillin-susceptible and -resistant S. aureus. The lugD gene was detected in 84% of the 56 S. lugdunensis isolates. All of the AP S. lugdunensis isolates (n = 23) carried the lugD gene and it was also detected in 24 of the non-AP isolates, suggesting different gene expression levels. One of the AP isolates stood out due to its high antimicrobial activity against more than 70% of the indicator bacteria tested, so it will be further characterized at genomic and proteomic level.

Bacteriocin-Like Inhibitory Substances in Staphylococci of Different Origins and Species With Activity Against Relevant Pathogens.

Bacteriocins are antimicrobial peptides with relevance in the modulation of human and animal microbiota that have gained interest in biomedical and biotechnological applications. In this study, the production of bacteriocin-like inhibitory substances (BLIS) was tested among a collection of 890 staphylococci of different origins (humans, animals, food, and the environment) and species, both coagulase-positive (CoPS, 238 isolates of 3 species) and coagulase-negative staphylococci (CoNS, 652 isolates of 26 species). Of the 890 staphylococci, 60 (6.7%) showed antimicrobial activity by the spot-on-lawn method against at least one of the 25 indicator bacteria tested. BLIS-producer (BLIS+) isolates were detected in 8.8% of CoPS and 6.0% of CoNS. The staphylococcal species with the highest percentages of BLIS+ isolates were S. chromogenes (38.5%), S. pseudintermedius (26.7%), and S. warneri (23.1%). The production of BLIS was more frequently detected among isolates of pets, wild animals, and food. Moreover, 13 BLIS+ isolates showed wide antimicrobial activiy spectrum, and 7 of these isolates (of species S. aureus, S. pseudintermedius, S. sciuri, and S. hominis) demonstrated antimicrobial activity against more than 70% of the indicator bacteria tested. The genetic characterization (by PCR and sequencing) of the 60 BLIS+ isolates revealed the detection of (a) 11 CoNS and CoPS isolates carrying putative lantibiotic-like genes; (b) 3 S. pseudintermedius isolates harboring the genes of BacSp222 bacteriocin; and (c) 2 S. chromogenes isolates that presented the gene of a putative cyclic bacteriocin (uberolysin-like), being the first report in this CoNS species. Antimicrobial susceptibility testing was performed in BLIS+ isolates and one-third of the CoNS isolates showed susceptibility to all antibiotics tested, which also lacked the virulence genes studied. These BLIS+ CoNS are good candidates for further characterization studies.

Beyond CC398: Characterisation of Other Tetracycline and Methicillin-Resistant Staphylococcus aureus Genetic Lineages Circulating in Spanish Hospitals.

Tetracycline resistance (TetR) has been evidenced as a good phenotypic marker for detection of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex CC398. The aim of this study was to characterise a collection of 95 TetR-MRSA isolates, not belonging to the lineage CC398, that were obtained in a previous multicentre study, to detect other MRSA clonal complexes that could be associated with this phenotypic TetR marker. The TetR-MRSA isolates were recovered from 20 Spanish hospitals during 2016 and they were characterised to determine their antimicrobial resistance and virulence phenotypes/genotypes as well as the presence of the immune evasion cluster (IEC). A high proportion of isolates belonging to the CC1 lineage (46%) were observed, as well as to the CC5, CC8 and CC45 lineages (11% each one). Thirty-two different spa-types were identified, being predominantly CC1-t127 (40%) and CC45-t1081 (11%). The IEC system (with the gene scn as marker) was present in 73% of isolates and 16% produced the Panton Valentine leucocidin (PVL). A high proportion of MRSA-CC1 isolates were scn-negative (38.6%) and 52.9% of them were blaZ-negative. A multidrug resistance (MDR) phenotype was identified in 86% of MRSA isolates. The knowledge of other TetR-MRSA genetic lineages, in addition to CC398, is highly relevant, since most of them were MDR and some of them presented important virulence factors. Strains potentially associated with livestock (as the subpopulation CC1-t127-scn-negative) or with humans (as the CC45 lineage or the subpopulation CC1-scn-positive) have been found in this study. The use of tetracycline-resistance for detection, not only of CC398 but also of other LA-MRSA lineages should be tracked in the future.

Ecology and Genetic Lineages of Nasal Staphylococcus aureus and MRSA Carriage in Healthy Persons with or without Animal-Related Occupational Risks of Colonization: A Review of Global Reports.

In this conceptual review, we thoroughly searched for appropriate English articles on nasal staphylococci carriage among healthy people with no reported risk of colonization (Group A), food handlers (Group B), veterinarians (Group C), and livestock farmers (Group D) published between 2000 and 2021. Random-effects analyses of proportions were performed to determine the pooled prevalence of S. aureus, MRSA, MRSA-CC398, and MSSA-CC398, as well as the prevalence of PVL-positive S. aureus from all eligible studies. A total of 166 eligible papers were evaluated for Groups A/B/C/D (n = 58/31/26/51). The pooled prevalence of S. aureus and MRSA in healthy humans of Groups A to D were 15.9, 7.8, 34.9, and 27.1%, and 0.8, 0.9, 8.6, and 13.5%, respectively. The pooled prevalence of MRSA-CC398 nasal carriage among healthy humans was as follows: Group A/B (<0.05%), Group C (1.4%), Group D (5.4%); and the following among Group D: pig farmers (8.4%) and dairy farmers (4.7%). The pooled prevalence of CC398 lineage among the MSSA and MRSA isolates from studies of the four groups were Group A (2.9 and 6.9%), B (1.5 and 0.0%), C (47.6% in MRSA), and D (11.5 and 58.8%). Moreover, MSSA-CC398 isolates of Groups A and B were mostly of spa-t571 (animal-independent clade), while those of Groups C and D were spa-t011 and t034. The MRSA-CC398 was predominately of t011 and t034 in all the groups (with few other spa-types, livestock-associated clades). The pooled prevalence of MSSA and MRSA isolates carrying the PVL encoding genes were 11.5 and 9.6% (ranges: 0.0-76.9 and 0.0-28.6%), respectively. Moreover, one PVL-positive MSSA-t011-CC398 isolate was detected in Group A. Contact with livestock and veterinary practice seems to increase the risk of carrying MRSA-CC398, but not in food handlers. Thus, this emphasizes the need for integrated molecular epidemiology of zoonotic staphylococci.

Airborne Dissemination of Bacteria (Enterococci, Staphylococci and Enterobacteriaceae) in a Modern Broiler Farm and Its Environment.

The role of the air as a vehicle of bacteria dissemination in the farming environment has been previously reported, but still scarcely studied. This study investigated the bacteria density/diversity of the inside and outside air and of litter samples at a broiler farm. Samples were collected considering two seasons, three outside air distances (50/100/150 m) and the four cardinal directions. Selective media was used for staphylococci, enterococci, and Enterobacteriaceae recovery. A high number of bacteria was detected in the litter (2.9 × 105-5.8 × 107 cfu/g) and in the inside air (>105 cfu/m3), but a low emission of bacteria was evidenced in the outside air (<6 cfu/m3). Moreover, the bacteria detected in the farm's outside air decreased the further from the farm the sample was taken. A total of 544 isolates were identified by MALDI-TOF (146 from the litter, 142 from inside air and 256 from outside air). From these, 162 staphylococci (14 species; S. saprophyticus 40.7%), 176 Enterobacteriaceae (4 species; E. coli 66%) and 190 enterococci (4 species; E. hirae 83%) were detected. E. hirae was the predominant species, and identical PFGE clones were detected in inside and outside samples. The detection of identical DNA profiles in E. hirae isolates from inside and outside samples suggests the role of the air in bacterial dissemination from the inside of the broiler farm to the immediate environment.

Penicillin susceptibility among invasive MSSA infections: a multicentre study in 16 Spanish hospitals.

OBJECTIVES: To determine the prevalence of penicillin susceptibility among MSSA causing bloodstream infections (BSIs) in 16 Spanish hospitals and to characterize the penicillin-susceptible MSSA (MSSA-PENS) isolates. METHODS: A total of 1011 Staphylococcus aureus isolates were collected from blood cultures in 16 Spanish hospitals during 2018-19 (6-12 months) and their susceptibility to 18 antimicrobials was determined. The MSSA-PENS isolates were selected and examined by PCR to determine the presence of the blaZ gene, other resistance genes and the genes lukF/lukS-PV, eta, etb and tst. The immune evasion cluster (IEC) type was also analysed. All the MSSA-PENS isolates were submitted to S. aureus protein A (spa) typing and the clonal complexes (CCs) were assigned according to their spa type. RESULTS: The prevalence of MSSA was 74.6% (754/1011) and 14.9% (151/1011) were MSSA-PENS-blaZnegative. MSSA-PENS-blaZnegative isolates (n = 151) were ascribed to 88 spa types and 11 CCs. The most frequent CCs were CC5 (35/151) and CC398 (25/151), with t002-CC5 and t571-CC398 being the most common lineages. Pan-susceptibility was identified in 117 of the 151 MSSA-PENS-blaZnegative isolates (77.5%). In the remaining isolates, erythromycin and clindamycin resistance was the most frequent resistance found, although tobramycin, ciprofloxacin, fusidic acid, mupirocin and/or tetracycline resistance was also detected. Thirty-eight MSSA-PENS-blaZnegative isolates were IEC negative and four isolates were Panton-Valentine leucocidin ('PVL') positive. CONCLUSIONS: A high penicillin susceptibility rate was detected among MSSA, opening therapeutic opportunities for BSIs. The emergence of new successful MSSA-PENS clones could be responsible for these data. The detection among MSSA-PENS-blaZnegative isolates of the clonal lineage CC398 or the absence of an IEC raises questions about their possible animal origin, requiring further analysis.

Genomic Analysis of Staphylococcus aureus of the Lineage CC130, Including mecC-Carrying MRSA and MSSA Isolates Recovered of Animal, Human, and Environmental Origins.

Most methicillin resistant Staphylococcus aureus (MRSA) isolates harboring mecC gene belong to clonal complex CC130. This lineage has traditionally been regarded as animal-associated as it lacks the human specific immune evasion cluster (IEC), and has been recovered from a broad range of animal hosts. Nevertheless, sporadic mecC-MRSA human infections have been reported, with evidence of zoonotic transmission in some cases. The objective of this study was to investigate the whole-genome sequences of 18 S. aureus CC130 isolates [13 methicillin-resistant (mecC-MRSA) and five methicillin-susceptible (MSSA)] from different sequences types, obtained from a variety of host species and origins (human, livestock, wild birds and mammals, and water), and from different geographic locations, in order to identify characteristic markers and genomic features. Antibiotic resistance genes found among MRSA-CC130 were those associated with the SSCmecXI element. Most MRSA-CC130 strains carried a similar virulence gene profile. Additionally, six MRSA-CC130 possessed scn-sak and one MSSA-ST130 had lukMF'. The MSSA-ST700 strains were most divergent in their resistance and virulence genes. The pan-genome analysis showed that 29 genes were present solely in MRSA-CC130 (associated with SCCmecXI) and 21 among MSSA-CC130 isolates (associated with phages). The SCCmecXI, PBP3, GdpP, and AcrB were identical at the amino acid level in all strains, but some differences were found in PBP1, PBP2, PBP4, and YjbH proteins. An examination of the host markers showed that the 3' region of the bacteriophage φ3 was nearly identical to the reference sequence. Truncated hlb gene was also found in scn-negative strains (two of them carrying sak-type gene). The dtlB gene of wild rabbit isolates included novel mutations. The vwbp gene was found in the three MSSA-ST700 strains from small ruminants and in one MSSA-ST130 from a red deer; these strains also carried a scn-type gene, different from the human and equine variants. Finally, a phylogenetic analysis showed that the three MSSA-ST700 strains and the two MSSA-ST130 strains cluster separately from the remaining MRSA-CC130 strains with the etD2 gene as marker for the main lineage. The presence of the human IEC cluster in some mecC-MRSA-CC130 strains suggests that these isolates may have had a human origin.

Prevalence and Genetic Characteristics of Staphylococcus aureus CC398 Isolates From Invasive Infections in Spanish Hospitals, Focusing on the Livestock-Independent CC398-MSSA Clade.

BACKGROUND: Livestock-associated (LA)-CC398-MRSA is closely related to pigs, being unfrequently detected in human invasive infections. CC398-MSSA is emerging in human invasive infections in some countries, but genetic and epidemiological characteristics are still scarcely reported. OBJECTIVES: To determine the prevalence of Staphylococcus aureus (SA) CC398, both MRSA and MSSA, among blood cultures SA isolates recovered in Spanish hospitals located in regions with different pig-farming densities (PD) and characterize the recovered isolates. METHODS: One thousand twenty-two SA isolates (761 MSSA, 261 MRSA) recovered from blood cultures during 6-12 months in 17 Spanish hospitals (2018-2019) were studied. CC398 lineage identification, detection of spa-types, and antibiotic resistance, virulence and human immune evasion cluster (IEC) genes were analyzed by PCR/sequencing. RESULTS: Forty-four CC398-MSSA isolates (4.3% of SA; 5.8% of MSSA) and 10 CC398-MRSA isolates (1% of SA; 3.8% of MRSA) were detected. Eleven spa-types were found among the CC398-MSSA isolates with t571 and t1451 the most frequent spa-types detected (75%). Most of CC398-MSSA isolates were Immune-Evasion-Cluster (IEC)-positive (88.6%), tetracycline-susceptible (95.5%) and erythromycin/clindamycin-inducible-resistant/erm(T)-positive (75%). No statistical significance was detected when the CC398-MSSA/MSSA rate was correlated to PD (pigs/km2) (p = 0.108). On the contrary, CC398-MRSA isolates were all IEC-negative, predominately spa-t011 (70%), and the CC398-MRSA/MRSA rate was significantly associated to PD (p < 0.005). CONCLUSION: CC398-MSSA is an emerging clade in invasive infections in Spanish hospitals. CC398-MRSA (mostly t011) and CC398-MSSA (mostly t571 and t1451) show important differences, possibly suggesting divergent steps in host-adaptation evolutionary processes. While CC398-MRSA is livestock-associated (lacking IEC-system), CC398-MSSA seems to be mostly livestock-independent, carrying human-adaptation markers.

Detection of a cfr-positive MRSA CC398 strain in a pig farmer in Spain / Detección de una cepa SARM del linaje CC398 y portadora del gen cfr en un granjero de cerdos en España

Objective: To characterize one linezolid- and methicillin-resistant Staphylococcus aureus (MRSA) isolate recovered from a nasal sample of a pig farmer patient. Methods: The detection of linezolid resistance mechanisms was performed by PCR and sequencing. The antimicrobial resistance and virulence profile was investigated, and the molecular typing was performed by molecular techniques. The transference of cfr gene was assessed by conjugation experiments and its genetic environment was investigated by specific PCRs. Results: The linezolid-resistant MRSA isolate was typed as t011-ST398/CC398-SCCmecV-agrI and carried the cfr gene. The isolate was multidrug-resistant but lacked the virulence genes studied. The cfr gene was co-located with the fexA gene on a Tn558 variant and was successfully transferred by conjugation. Conclusion: We report the first description of LA-MRSA-CC398 carrying the cfr gene in Spain. This finding highlights the importance of surveillance programmes to determine the presence and spread of the cfr gene in the livestock and clinical settings.(AU)

Objetivo: Caracterizar una cepa Staphylococcus aureus resistente a meticilina (SARM) y a linezolid aislada de una muestra nasal de un paciente granjero de cerdos. Métodos: Mediante PCR y secuenciación se investigaron los mecanismos de resistencia a linezolid. Se determinó el perfil de resistencia a antimicrobianos y el perfil de virulencia, y se llevó a cabo el tipado molecular mediante diferentes técnicas moleculares. Se estudió la transferibilidad del gen cfr por conjugación y su entorno genético mediante PCR específicas. Resultados: El aislado fue tipado como t011-ST398/CC398-SCCmecV-agrI y portaba el gen cfr. Presentó un feno/genotipo de multirresistencia, pero carecía de los genes de virulencia estudiados. Se detectó el gen cfr junto con fexA en una variante del Tn558 y se transfirió mediante conjugación. Conclusión: Se describe el primer aislado SARM-CC398 cfr-positivo en España. Esto destaca la importancia de implementar programas de vigilancia para determinar su presencia y dispersión en el ámbito clínico y ganadero.(AU)

Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa, Algeria.

This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.

S. pseudintermedius and S. aureus lineages with transmission ability circulate as causative agents of infections in pets for years.

BACKGROUND: Staphylococcus pseudintermedius (SP) and Staphylococcus aureus (SA) are common colonizers of companion animals, but they are also considered opportunistic pathogens, causing diseases of diverse severity. This study focused on the identification and characterization of 33 coagulase-positive staphylococci isolated from diseased pets (28 dogs and five cats) during 2009-2011 in a veterinary hospital in Spain in order to stablish the circulating lineages and their antimicrobial resistance profile. RESULTS: Twenty-eight isolates were identified as SP and five as SA. Nine methicillin-resistant (MR) isolates (27%) carrying the mecA gene were detected (eight MRSP and one MRSA). The 55% of SP and SA isolates were multidrug-resistant (MDR). MRSP strains were typed as ST71-agrIII-SCCmecII/III-(PFGE) A (n=5), ST68-agrIV-SCCmecV-B1/B2 (n=2), and ST258-agrII-SCCmecIV-C (n=1). SP isolates showed resistance to the following antimicrobials [percentage of resistant isolates/resistance genes]: penicillin [82/blaZ], oxacillin [29/mecA] erythromycin/clindamycin [43/erm(B)], aminoglycosides [18-46/aacA-aphD, aphA3, aadE], tetracycline [71/tet(M), tet(K)], ciprofloxacin [29], chloramphenicol [29/catpC221], and trimethoprim-sulfamethoxazole [50/dfrG, dfrK]. The dfrK gene was revealed as part of the radC-integrated Tn559 in two SP isolates. Virulence genes detected among SP isolates were as follow [percentage of isolates]: siet [100], se-int [100], lukS/F-I [100], seccanine [7], and expB [7]. The single MRSA-mecA detected was typed as t011-ST398/CC398-agrI-SCCmecV and was MDR. The methicillin-susceptible SA isolates were typed as t045-ST5/CC5 (n=2), t10576-ST1660 (n=1), and t005-ST22/CC22 (n=1); the t005-ST22 feline isolate was PVL-positive and the two t045-ST45 isolates were ascribed to Immune Evasion Cluster (IEC) type F. Moreover, the t10576-ST1660 isolate, of potential equine origin, harbored the lukPQ and scneq genes. According to animal clinical history and data records, several strains seem to have been acquired from different sources of the hospital environment, while some SA strains appeared to have a human origin. CONCLUSIONS: The frequent detection of MR and MDR isolates among clinical SP and SA strains with noticeable virulence traits is of veterinary concern, implying limited treatment options available. This is the first description of MRSA-ST398 and MRSP-ST68 in pets in Spain, as well the first report of the dfrK-carrying Tn559 in SP. This evidences that current transmissible lineages with mobilizable resistomes have been circulating as causative agents of infections among pets for years.

Extended Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae from Broiler Liver in the Center of Algeria, with Detection of CTX-M-55 and B2/ST131-CTX-M-15 in Escherichia coli.

This study aimed to determine the prevalence and diversity of extended-spectrum ß-lactamase (ESBL)-producing and multidrug-resistant (MDR) Escherichia coli and Klebsiella pneumoniae isolates from 136 broiler livers randomly purchased in 136 retail markets in Djelfa (Algeria). Isolation was performed on Hektoen agar and bacterial identification was carried out by API20E system and Maldi-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Antimicrobial susceptibility was tested by the disk diffusion and agar dilution methods. Detection of ESBLs and other resistance and integron genes, phylogenetic grouping, and molecular typing was performed by PCR and sequencing. Seventy-eight isolates (one per positive sample) were recovered: 73 E. coli and 5 K. pneumoniae. Among E. coli, 86.3% of isolates were MDR. ESBL activity was revealed in eight E. coli and five K. pneumoniae isolates (rates of 5.9% and 3.7% in analyzed samples, respectively). ESBL genes detected among E. coli were as follows (number of isolates): blaCTX-M-15 (3), blaCTX-M-1 (3), blaCTX-M-55 (1), and blaSHV-12 (1); all ESBL-producing K. pneumoniae isolates carried the blaCTX-M-15 gene. ESBL-producing E. coli isolates were assigned to lineages (phylogroup/sequence type and number of isolates in parenthesis): A/ST48 (1), B1/ST6448 (1), B1/ST5087 (3), B1/ST23 (1), and B2/ST131 (two blaCTX-M-15 E. coli isolates). K. pneumoniae isolates were ascribed to sequence types ST2010 and ST3483. Regarding the 65 non-ESBL E. coli isolates, the most observed resistance genes were as follows: tet(A) (75%), blaTEM (57.1%), and sul2 (43.5%). Class1 integrons were revealed in seven non-ESBL E. coli isolates (10.7%) and two gene-cassette arrays were identified: dfrA1 and aadA1+dfrA1. Our study provides evidence that broiler-derived food from Center of Algeria constitutes a source of ESBL and/or MDR-producing Enterobacteriaceae, with detection of relevant ESBL genes and epidemic clones.

Detection of a cfr-positive MRSA CC398 strain in a pig farmer in Spain.

OBJECTIVE: To characterize one linezolid- and methicillin-resistant Staphylococcus aureus (MRSA) isolate recovered from a nasal sample of a pig farmer patient. METHODS: The detection of linezolid resistance mechanisms was performed by PCR and sequencing. The antimicrobial resistance and virulence profile was investigated, and the molecular typing was performed by molecular techniques. The transference of cfr gene was assessed by conjugation experiments and its genetic environment was investigated by specific PCRs. RESULTS: The linezolid-resistant MRSA isolate was typed as t011-ST398/CC398-SCCmecV-agrI and carried the cfr gene. The isolate was multidrug-resistant but lacked the virulence genes studied. The cfr gene was co-located with the fexA gene on a Tn558 variant and was successfully transferred by conjugation. CONCLUSION: We report the first description of LA-MRSA-CC398 carrying the cfr gene in Spain. This finding highlights the importance of surveillance programmes to determine the presence and spread of the cfr gene in the livestock and clinical settings.

Mechanisms of Linezolid Resistance Among Clinical Staphylococcus spp. in Spain: Spread of Methicillin- and Linezolid-Resistant S. epidermidis ST2.

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical Staphylococcus aureus (n = 2) and coagulase-negative staphylococci (n = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the cfr gene was determined by whole-genome sequencing. The cfr gene was detected in one methicillin-resistant S. aureus (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second S. aureus isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant Staphylococcus epidermidis (n = 14) and Staphylococcus hominis isolates (n = 1) showed the mutation G2576T (n = 14) or C2534T (n = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in S. epidermidis isolates. All S. epidermidis isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the mecA gene. The cfr gene in the MRSA isolate was located together with the fexA gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant S. epidermidis ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected.

Genetic characterization of extended-spectrum ß-lactamase-producing Enterobacteriaceae from a biological industrial wastewater treatment plant in Tunisia with detection of the colistin-resistance mcr-1 gene.

This study evaluated the occurrence of extended-spectrum ß-lactamases (ESBL) and associated resistance genes, integrons, and plasmid types, as well as the genetic relatedness of enterobacterial isolates in the wastewater treatment plant (WWTP) of La Charguia, Tunis City (Tunisia). A total of 100 water samples were collected at different points of the sewage treatment process during 2017-2019. Antimicrobial susceptibility was conducted by the disc-diffusion method. blaCTX-M, blaTEM and blaSHV genes as well as those encoding non-ß-lactam resistance, the plasmid types, occurrence of class1 integrons and phylogenetic groups of Escherichia coli isolates were determined by PCR/sequencing. Genomic relatedness was determined by multi-locus sequence typing (MLST) for selected isolates. In total, 57 ESBL-producer isolates were recovered (47 E. coli, eight Klebsiella pneumoniae, 1 of the Citrobacter freundii complex and 1 of the Enterobacter cloacae complex). The CTX-M-15 enzyme was the most frequently detected ESBL, followed by CTX-M-27, CTX-M-55 and SHV-12. One E. coli isolate harboured the mcr-1 gene. The following phylogroups/sequence types (STs) were identified among ESBL-producing E. coli isolates: B2/ST131 (subclade-C1), A/ST3221, A/ST8900, D/ST69, D/ST2142, D/ST38, B1/ST2460 and B1/ST6448. High numbers of isolates harboured the class 1 integrons with various gene cassette arrays as well as IncP-1 and IncFIB plasmids. Our findings confirm the importance of WWTPs as hotspot collectors of ESBL-producing Enterobacteriaceae with a high likelihood of spread to human and natural environments.

Bacteriemia por Staphylococcus aureus sensible a penicilina. Importancia epidemiológica, clínica y posibles implicaciones terapéuticas / Penicillin-susceptible Staphylococcus aureus bacteremia: Epidemiological and clinical relevance. Possible therapeutic implications

INTRODUCCIÓN: El aumento de la sensibilidad a penicilina en Staphylococcus aureus (SA-PenS) podría tener relevancia terapéutica. Pretendemos conocer esta situación en nuestro medio. MATERIAL Y MÉTODOS: Se analizaron bacteriemias por SA en un hospital durante 2,5 años (2015-2017). Estudiamos la sensibilidad a antimicrobianos, genes de resistencia a beta-lactámicos (blaZ, mecA) y presencia de leucocidina de Panton-Valentine. En aislados SA-PenS-blaZnegativo se determinó el tipo de spa, MLST y genes de resistencia a antimicrobianos no-beta-lactámicos. RESULTADOS: Hubo 84 pacientes con bacteriemia por SA (35,7% SARM y 64,3% SASM), se analizaron 77. El 22% de los SASM estudiados (n = 11) fueron PenS-blaZnegativo (CMI-Pen ≤ 0,3 μg/ml), correspondiendo al 14,3% del total de SA. En SASM-PenS-blaZnegativo se detectaron 8 tipos-spa y 7 complejos clonales. CONCLUSIÓN: Detectamos alta prevalencia de SARM/SA y de SASM-PenS-blaZnegativo/SASM en hemocultivos. Una CMI-Pen ≤ 0,3 μg/ml se correspondió con SASM-PenS-blaZnegativo. Esta situación plantea opciones terapéuticas que deberán reevaluarse con estudios más amplios y ensayos clínicos

INTRODUCTION: The increase in penicillin susceptibility among Staphylococcus aureus (SA-PenS) might have therapeutic relevance. We aimed to study the current situation in our environment. MATERIAL AND METHODS: Over a 2.5 years period, all SA isolates from bacteraemia in one hospital were analysed. For all isolates, antimicrobial susceptibility profile, beta-lactam resistance genes (blaZ, mecA) and Panton-Valentine leucocidine encoding-genes were studied. For SA-PenS-blaZnegative isolates, spa-type, MLST and the presence of other resistance genes were studied. RESULTS: Among 84 patients with SA bacteraemia (35.7% MRSA and 64.3% MSSA), 77 were analysed; 22.2% of MSSA isolates were PenS and blaZnegative (Pen-MIC ≤ 0.03 μg/ml) corresponding to 14.3% of the total SA. In MSSA-PenS-blaZnegative isolates, eight spa-types and 7 clonal-complexes were detected. CONCLUSION: A high prevalence of MRSA/SA and MSSA-PenS-blaZnegative/MSSA was detected in blood cultures. Pen-MIC ≤ 0,3 μg/ml corresponded to MSSA-PenS-blaZnegative. This situation raises therapeutic options which should be further evaluated in larger studies and clinical trials

Human mecC-Carrying MRSA: Clinical Implications and Risk Factors.

A new methicillin resistance gene, named mecC, was first described in 2011 in both humans and animals. Since then, this gene has been detected in different production and free-living animals and as an agent causing infections in some humans. The possible impact that these isolates can have in clinical settings remains unknown. The current available information about mecC-carrying methicillin resistant S. aureus (MRSA) isolates obtained from human samples was analyzed in order to establish its possible clinical implications as well as to determine the infection types associated with this resistance mechanism, the characteristics of these mecC-carrying isolates, their possible relation with animals and the presence of other risk factors. Until now, most human mecC-MRSA infections have been reported in Europe and mecC-MRSA isolates have been identified belonging to a small number of clonal complexes. Although the prevalence of mecC-MRSA human infections is very low and isolates usually contain few resistance (except for beta-lactams) and virulence genes, first isolates harboring important virulence genes or that are resistant to non-beta lactams have already been described. Moreover, severe and even fatal human infection cases have been detected. mecC-carrying MRSA should be taken into consideration in hospital, veterinary and food safety laboratories and in prevention strategies in order to avoid possible emerging health problems.

Importancia del complejo clonal CC398 en las bacteriemias por Staphylococcus aureus en un hospital secundario de Aragón / Relevance of clonal complex CC398 in bacteremia caused by Staphylococcus aureus in a secondary hospital of Aragon, Spain

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