Genomic insights into the expansion of carbapenem-resistant Klebsiella pneumoniae within Portuguese hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal, the CR-KP rate has increased sharply, but the factors associated with this increase are poorly explored. In order to address this question, phylogenetic and resistome analysis were used to compare the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. Most CR-KP belonged to sequence type (ST) 13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by the presence of KPC-3 (74%) or OXA-181 (18%), which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multi-drug resistant, harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infecting isolates were highly related, and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified by core genome multi-locus sequence typing data analysis (fewer than five allelic differences), 41 occurred between different hospitals and 130 occurred within the same hospital. The results suggest that CR-KP dissemination in the Lisbon region results from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients, and transmission of CR-KP within and between hospitals. Prudent use of carbapenems, patient screening at hospital entry, and improvement of infection control are needed to decrease the burden of CR-KP infection in Portugal.

Population structure of methicillin-susceptible Staphylococcus aureus (MSSA) in Portugal over a 19-year period (1992-2011).

Despite their clinical relevance, few studies have addressed the epidemiology of methicillin-susceptible S. aureus (MSSA). In particular, it is not clear how MSSA population structure has evolved over time and how it might have been shaped by the emergence of MRSA in the community (CA-MRSA). In the present study we have evaluated the MSSA population structure over time, its geographical distribution and relatedness with MRSA in Portugal. A total of 465 MSSA from infection and colonization, collected over a 19-year period (1992-2011) in the northern, central and southern regions of Portugal were analyzed. Isolates were characterized by spa typing and multilocus-sequence typing (MLST). Isolates with predominant spa types were characterized by pulsed-field gel electrophoresis (PFGE). Isolates relatedness was analyzed by eBURST and BURP. The 172 spa types found among the 465 MSSA were grouped into 18 spa-CC (clonal complexes). Ten clonal types were more prevalent (40 %): one major clone (ST30-t012) was present in the entire study period and all over the country and the other nine were intermittently detected over time (ST5-t002, ST8-t008, ST15-t084, ST34-t166, ST72-t148, ST1-t127, ST7-t091, ST398-t571 and ST34-t136). Interestingly, three MSSA clonal types observed only after 1996 were closely related with CA-MRSA epidemic strains (ST8-t008, ST72-t148 and ST1-t127) found currently in Portugal. The MSSA population in Portugal is genetically diverse; however, some dominant clonal types have been established and widely disseminated for almost two decades. We identified MSSA isolates that were related with emergent CA-MRSA clones found in Portugal.

High prevalence of hospital-associated methicillin-resistant Staphylococcus aureus in the community in Portugal: evidence for the blurring of community-hospital boundaries.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infection in the community (CA-MRSA), but in spite of its relevance, no data exist concerning its epidemiology in Portugal. In this study, we aimed to evaluate the prevalence, population structure, and origin of MRSA in the Portuguese community. A total of 527 isolates, both methicillin-susceptible S. aureus (MSSA) and MRSA, were collected from individuals with no healthcare-related risk factors attending 16 healthcare institutions in Portugal. Isolates were characterized for the presence of mecA, Panton-Valentine leukocidin (PVL), and arginine catabolic mobile element (ACME), and by staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa, and multilocus sequence typing (MLST). Susceptibility to a panel of 13 antibiotics was tested. Isolates relatedness was analyzed by goeBURST and BURP. We found a high frequency (21.6%) of MRSA in the community. However, only 11.4% of the isolates belonged to typical CA-MRSA epidemic clones (USA300, USA400, USA700, Southwest Pacific, European, and ST398). The remaining isolates, which constituted the great majority (88.6%), belonged to hospital-associated MRSA (HA-MRSA) epidemic clones, namely, to the EMRSA-15 clone (77.2%). PVL was rare and carried by 17 isolates only (five MRSA and 12 MSSA). In the whole collection, some MRSA and MSSA were highly related. The high frequency of MRSA in the community in Portugal seems to result mainly from dissemination from the hospital. They might also have emerged from an extant MSSA population, by SCCmec acquisition, or MRSA clonal introduction from abroad.

High prevalence of mec complex C and ccrC is independent of SCCmec type V in Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most clinically relevant coagulase-negative staphylococci (CoNS), particularly in immunocompromised patients; however, little is known regarding its molecular epidemiology. In this work, we characterized the genetic background and the SCCmec region of 36 methicillin-resistant S. haemolyticus (MRSHae) and 10 methicillin-susceptible S. haemolyticus (MSSHae) collected from neutropenic patients in Tunisia between 2002 and 2004. The molecular characterization of MRSHae by pulsed-field gel electrophoresis (PFGE) showed that the great majority of the isolates (77.8%) belonged to only four types. SCCmec typing by polymerase chain reaction (PCR) and Southern hybridization showed that isolates belonging to each PFGE type could carry either one or two SCCmec types. SCCmec V was the most common, but mec complex C was frequently associated to ccr allotypes other than ccrC. The mec complex class C was predominant in MRSHae (47%) and ccrC was predominant among both methicillin-resistant and -susceptible isolates (31 and 50%, respectively). Interestingly, one half (50%) of the MRSHae isolates analyzed lacked the known ccr complexes (ccrAB and ccrC), although they carried the mecA. Conversely, all MSSHae carrying a ccrC complex were multidrug-resistant, although they lack the mecA. The results suggest that ccrC and mec complex C are frequent and may exist autonomously and independently of SCCmec type V in S. haemolyticus. Moreover, the data obtained suggest that small chromosomal rearrangements promoting the loss or structural variation of mec and ccr complex appear to occur frequently, which probably provide S. haemolyticus with a specialized means for SCCmec trapping and/or diversification.

Community-associated methicillin-resistant Staphylococcus aureus infection in Portugal.

Methicillin-resistant Staphylococcus aureus (MRSA) has recently emerged as a cause of community-acquired infections among individuals without risk factors. Community-associated MRSA (CA-MRSA) appears to be more virulent, causing superficial mild skin and soft tissue infections to severe necrotizing fasciitis, and in rare cases, pneumonia. Community-associated MRSA was first reported in Australia in the early 80s, after almost two decades in the USA, and then in several countries in Europe, Asia and South America. No data exists in Portugal. We report the first case of CA-MRSA infection in Portugal, in a young adult with severe necrotizing pneumonia, complicated with bilateral empyema and respiratory failure.

Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI.

In order to obtain insights into the methicillin-resistant Staphylococcus aureus (MRSA) population structure in the Azores archipelago, 106 MRSA isolates were collected from patients attending an Azorean central hospital between January 2007 and February 2008. Antimicrobial resistance was determined for all isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec) typing and the presence of Panton-Valentine leukocidin (PVL). The majority of the isolates (87%, n = 92) belonged to the EMRSA-15 clone (ST22, SCCmec-IVh), followed by the Pediatric clone (ST5-VI/IVc) (11%, n = 12). The Berlin clone (ST45-IVa) and a new clone (spa type t1839, ST1339 and SCCmec V variant) were represented by single isolates. All of the isolates carried SCCmec types IV, V or VI and a non-multiresistant antibiotic profile, resembling the currently emerging community MRSA. Moreover, PVL was described for the first time to be associated with the Pediatric clone carrying SCCmec type VI. We provided the first description of the population structure of MRSA in the Azores islands, which seems to be shaped by genetic events occurring locally, as well as by the regular population exchange between the islands, continental Portugal, the United Kingdom and the United States.

Comparison of molecular typing methods for characterization of Staphylococcus epidermidis: proposal for clone definition.

In the present study we give some direction on the selection of the most appropriate typing method(s) to be used for the characterization of Staphylococcus epidermidis, in view of the most recent findings on the evolution, population structure, and epidemiology of this species. In order to achieve this aim, quantitative assessment of the correlation of the results of three typing methods--pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing, which target different regions of the chromosome that evolve at different rates--was performed. In order to evaluate the discriminatory ability and the strength and direction of the correlation of the different typing methods, Simpson's index of diversity (SID), the adjusted Rand coefficient (AR), and the Wallace coefficient (W) were calculated. PFGE was the most discriminatory method (SID = 99%), followed by MLST (SID = 90%) and SCCmec typing (SID = 75%). The values of AR and W (0.10 < AR < 0.30; 0.50 < W < 0.75) indicated that the partition of the same isolate collection by PFGE, MLST, and SCCmec typing provided results that had only a poor correlation with each other. However, the information provided by the combination of PFGE and SCCmec enabled the prediction of the results obtained by MLST at the level of the clonal complex with a high degree of precision (W > 0.90). We propose that clones of S. epidermidis be defined by the combination of the PFGE type followed by the SCCmec type, which provides reliable information on the short-term epidemiology and the ability to predict with consistency long-term clonal evolution.

Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data.

Despite its importance as a human pathogen, information on population structure and global epidemiology of Staphylococcus epidermidis is scarce and the relative importance of the mechanisms contributing to clonal diversification is unknown. In this study, we addressed these issues by analyzing a representative collection of S. epidermidis isolates from diverse geographic and clinical origins using multilocus sequence typing (MLST). Additionally, we characterized the mobile element (SCCmec) carrying the genetic determinant of methicillin resistance. The 217 S. epidermidis isolates from our collection were split by MLST into 74 types, suggesting a high level of genetic diversity. Analysis of MLST data using the eBURST algorithm revealed the existence of nine epidemic clonal lineages that were disseminated worldwide. One single clonal lineage (clonal complex 2) comprised 74% of the isolates, whereas the remaining isolates were clustered into 8 minor clonal lineages and 13 singletons. According to our evolutionary model, SCCmec was acquired at least 56 times by S. epidermidis. Although geographic dissemination of S. epidermidis strains and the value of the index of association between the alleles, 0.2898 (P < 0.05), support the clonality of S. epidermidis species, examination of the sequence changes at MLST loci during clonal diversification showed that recombination gives rise to new alleles approximately twice as frequently as point mutations. We suggest that S. epidermidis has a population with an epidemic structure, in which nine clones have emerged upon a recombining background and evolved quickly through frequent transfer of genetic mobile elements, including SCCmec.

Genetic diversity among methicillin-resistant Staphylococcus epidermidis (MRSE).

We selected 106 methicillin-resistant Staphylococcus epidermidis (MRSE) and 22 methicillin-susceptible S. epidermidis (MSSE) hospital isolates--each with a different PFGE pattern--for more detailed documentation of genetic diversity. The 106 MRSE isolates showed extensive variation in the SmaI DNA fragments hybridizing with the DNA probe for mecA, the molecular size of which varied from as low as 20 kb up to over 500 kb. Parallel variation was also observed in the size of DNA fragments hybridizing with the chromosomal genes orfX and gyrA, and this was also observed in MSSE isolates. In contrast, SmaI fragments associated with the housekeeping genes murE and aroE, both located distantly from orfX, showed little size variation. Typing for the mec complex and ccr identified 10 different SCCmec structures and a large number of strains (21 isolates) that were non-typeable. The majority of strains studied (36%) carried a SCCmec type IV-like structure, including strains with non-related PFGE profiles. On the other hand, closely related strains often carried different types of SCCmec. The findings indicate that the acquisition and/or loss of mobile genetic elements, including various structural types of SCCmec, may occur frequently in the vicinity of the orfX gene on the S. epidermidis chromosome.

Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR.

The emergence of coagulase-negative staphylococci not only as human pathogens but also as reservoirs of antibiotic resistance determinants requires the deployment and development of methods for their rapid and reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was used to identify a collection of 617 clinical staphylococcal isolates. The amplicons were resolved in high-resolution agarose gels and visually compared with the patterns obtained for the control strains of 29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%) were identified by ITS-PCR and included 11 species: 302 isolates of Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had unique ITS-PCR patterns, although some were very similar, namely, the group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus, S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species, S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a valuable alternative for the identification of staphylococci, offering, within the same response time and at lower cost, higher reliability than the currently available commercial systems.

Three-year assessment of methicillin-resistant Staphylococcus aureus clones in Latin America from 1996 to 1998.

Four hundred ninety-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America-Argentina, Brazil, Chile, Uruguay and Mexico-were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal SmaI digests (ClaI-mecA::ClaI-Tn554::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::kappa::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E'::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.