Paulo Martins da Costa

Transfer of Multidrug-Resistant Bacteria Between Intermingled Ecological Niches: The Interface Between Humans, Animals and the Environment

The use of antimicrobial agents has been claimed to be the driving force for the emergence and spread of microbial resistance. However, several studies have reported the presence of multidrug-resistant bacteria in populations exposed to low levels of antimicrobial drugs or even never exposed. For many pathogens, especially those organisms for which asymptomatic colonization typically precedes infection (e.g., Enterococcus spp. and Escherichia coli), the selective effects of antimicrobial use can only be understood if we considerer all biological and environmental pathways which enable these bacteria, and the genes they carry, to spread between different biomes. This ecological framework provides an essential perspective for formulating antimicrobial use policies, precisely because it encompasses the root causes of these problems rather than merely their consequences.

Seagulls and Beaches as Reservoirs for Multidrug-Resistant Escherichia coli

A variety of extended-spectrum β-lactamase–producing Escherichia coli isolates, with a high rate of cefotaximase-15 resistance, were identified in seagull feces from Porto, Portugal, beaches. Beaches may therefore present a risk to public health because of the potential pathogen-spreading capacity of migratory birds.

High Prevalence of EMRSA-15 in Portuguese Public Buses: A Worrisome Finding

Background The nosocomial prevalence of methicillin resistant Staphylococcus aureus (MRSA) in Portugal remains one of the highest in Europe and is currently around 50%. Transmission of S. aureus, including MRSA, occurs principally by direct human-to-human skin contact. However, S. aureus can survive for long periods on inanimate objects, which may represent an important reservoir for dissemination as well. Methodology/Principal Findings Between May 2009 and February 2010, handrails of 85 public urban buses circulating in Oporto, Portugal, were screened for the occurrence of MRSA. Twenty-two (26%) buses showed MRSA contamination. The molecular characterization of a total of 55 MRSA, by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome (SCC) mec typing, spa typing, and multilocus sequence typing (MLST), clustered the isolates into three clonal types. However, the overwhelming majority (n = 50; 91%) of the isolates belonged to a single clone (PFGE A, spa types t747, t032, t025 or t020, ST22, SCCmec type IVh) that exhibits the characteristics of the pandemic EMRSA-15, currently the major lineage circulating in Portuguese hospitals, namely in the Oporto region. Two additional clones were found but in much lower numbers: (i) PFGE B, ST5, spa type t002, SCCmec IVa (n = 3), and (ii) PFGE C, spa type t008, ST8, SCCmec IVa (n = 2). None of the 55 isolates was PVL positive. Conclusions/Significance Public buses in Oporto seem to be an important reservoir of MRSA of nosocomial origin, providing evidence that the major hospital-associated MRSA clone in Portugal is escaping from the primary ecological niche of hospitals to the community environment. Infection control measures are urgently warranted to limit the spread of EMRSA-15 to the general population and future studies are required to assess the eventual increase of MRSA in the Portuguese community, which so far remains low.

Genetic Detection of Extended-Spectrum β-Lactamase-Containing Escherichia coli Isolates from Birds of Prey from Serra da Estrela Natural Reserve in Portugal

ABSTRACT Extended-spectrum β-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following β-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).

Vancomycin‐resistant enterococci from Portuguese wastewater treatment plants

The objective of this study was to evaluate the incidence of vancomycin resistant enterococci in sludge and sewage of urban and poultry‐slaughterhouse wastewater treatment plants. A total of 17 vancomycin resistant enterococci (eight vanA ‐containing Enterococcus faecium and nine vanC1/vanC2 ‐containing Enterococcus gallinarum/casseliflavus) were found among 499 isolates of sewage and sludge samples of 14 urban and nine poultry‐slaughterhouse wastewater treatment plants. These seventeen VRE isolates showed resistance to kanamycin (n = 8), tetracycline (n = 7), erythromycin (n = 7), ciprofloxacin (n = 7), ampicillin (n = 7), streptomycin (n = 6), and gentamicin (n = 2). The tetM gene, related with tetracycline resistance, was found in six of eight van A‐containing isolates, in all seven vanC‐1 isolates and in one of two vanC‐2 isolates. The ermB gene in seven erythromycin‐resistant isolates; and the aac6 ′‐aph2 ″ gene in the two high‐level‐gentamicin‐resistant isolates. Moreover, two vanA ‐containing E. faecium isolates harbored the hyl virulence gene, and three isolates the entA bacteriocin gene. The purK‐1 allele was detected in our urban vanA ‐containing E. faecium isolate, and we found as well the purK‐6 allele in one poultry‐slaughterhouse vanA ‐containing E. faecium isolate. This study suggests that the wastewater treatment plants might be an important source of dissemination of antibiotic‐resistant enterococci in Portugal. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Antibacterial and Antibiofilm Activities of Tryptoquivalines and Meroditerpenes Isolated from the Marine-Derived Fungi Neosartorya paulistensis, N. laciniosa, N. tsunodae, and the Soil Fungi N. fischeri and N. siamensis

A new meroditerpene, sartorypyrone C (5), was isolated, together with the known tryptoquivalines l (1a), H (1b), F (1c), 3′-(4-oxoquinazolin-3-yl) spiro[1H-indole-3,5′]-2,2′-dione (2) and 4(3H)-quinazolinone (3), from the culture of the marine sponge-associated fungus Neosartorya paulistensis (KUFC 7897), while reexamination of the fractions remaining from a previous study of the culture of the diseased coral-derived fungus N. laciniosa (KUFC 7896) led to isolation of a new tryptoquivaline derivative tryptoquivaline T (1d). Compounds 1a–d, 2, 3, and 5, together with aszonapyrones A (4a) and B (4b), chevalones B (6) and C (7a), sartorypyrones B (7b) and A (8), were tested for their antibacterial activity against four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa), as well as the environmental multidrug-resistant isolates. Only aszonapyrone A (4a) and sartorypyrone A (8) exhibited significant antibacterial activity as well as synergism with antibiotics against the Gram-positive multidrug-resistant strains. Antibiofilm assays of aszonapyrone A (4a) and sartorypyrone A (8) showed that practically no biofilm was formed in the presence of their 2× MIC and MIC. However, the presence of a sub-inhibitory concentration of ½ MIC of 4a and 8 was found to increase the biofilm production in both reference strain and the multidrug-resistant isolates of S. aureus.

Environmental KPC-Producing Escherichia coli Isolates in Portugal

Carbapenemase-producing isolates of the Enterobacteriaceae are reported increasingly worldwide (9). Besides the emergence of OXA-48 and NDM-1 producers in specific geographical areas, KPC-producing isolates are endemic in many places (8). Those KPCs hydrolyze all -lactams, including carbapenems at a significant level, with the exception of cephamycins. The blaKPClike genes have been reported most often for Klebsiella pneumoniae, but they have been additionally reported repeatedly for other enterobacterial species. Moreover, some KPC-producing Pseudomonas aeruginosa and Acinetobacter baumannii isolates have been reported (8, 9). Besides the United States, KPCproducing enterobacterial isolates are at least endemic in Columbia, Greece, and Italy (9). Our study was conducted in order to evaluate whether the aquatic environment in Portugal could be a reservoir of carbapenem-resistant enterobacterial isolates. Five water samples were collected in December 2010 at different locations from one river crossing the city of Santo Tirso, north Portugal. Samples of 100 ml (each) were filtered on a 0.45m sterile filter, and the corresponding filter was placed on an imipenem (1 g/ml)containing Drigalski plate. Only one type of colony grew, corresponding to Escherichia coli. MICs of E. coli strain MAS were determined by the Etest method (AB bioMérieux, Solna, Sweden) and interpreted according to updated CLSI breakpoints (2). It was resistant to all -lactams, including to all carbapenems (Table 1). That strain remained susceptible only to tetracycline, fosfomycin, and colistin, being resistant to all fluoroquinolones and aminoglycosides (Table 1). Molecular investigations were then performed using PCR in order to search for carbapenemase genes, followed by sequencing (6). This allowed the identification of the blaKPC-2 -lactamase gene. Analysis of the plasmid content of E. coli isolate MAS identified a single plasmid of ca. 150 kb that was successfully transferred to E. coli J53 by conjugation, with selection performed on amoxicillin (100 g/ml) and azide (100 g/ml)-containing agar plates (6). The blaKPC-2-positive plasmid was identified as an IncF plasmid by using PCR-based replicon typing (1). It cotransferred reduced susceptibility to gentamicin and amikacin. PCR mapping performed as described previously (6) showed that the blaKPC-2 gene was part of transposon Tn4401a. Multilocus sequence typing (MLST) performed according to the protocol described on the E. coli MLST website (http://www.pasteur .fr/recherche/genopole/PF8/mlst/EColi.html) showed that E. coli MAS belonged to the ST410 type. Further samplings were obtained in June 2011 at the same place, and selection was performed under the same conditions, but no carbapenem-nonsusceptible E. coli grew. This is the first identification of a KPC-producing E. coli in Portugal. It is noteworthy that the blaKPC-2 gene was identified in E. coli, in which it has been rarely found, with only a few reports from the United States, Israel, Brazil, and France (3–5, 7). Surprisingly, it has been recovered from an environmental sample, whereas no human case has been reported so far, corresponding therefore to the very first identification of KPC in this environment. To explain those findings, we might speculate that some people living in the neighboring area could spread these KPC producers in the environment. Another possibility is that the aquatic environment could actually contain KPC producers, suggesting that it might represent the source of a future human colonization. Of particular interest is the identification of the strain as belonging to the ST410 type, considering that extended-spectrum -lactamase (ESBL)-producing ST410 E. coli was recently identified in Brazil, where KPC enzymes are widespread (10). Based on the close relationship between Portugal and Brazil in terms of population exchange, it could therefore be speculated that a link might exist.

Effects of antimicrobial treatment on selection of resistant Escherichia coli in broiler fecal flora.

Under field conditions, three commercial antimicrobials were sequentially prescribed to 16,000 broiler chickens during their rearing period, via drinking water using subtherapeutic levels for 3 days. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Feed diet and fecal samples from both groups were collected periodically. One sample of the drinking water along with samples from the broiler house environment was also collected 1 day before bird placement. Samples were plated onto Tergitol BCIG Agar media; a maximum of 26 Escherichia coli were isolated per sample, and their susceptibility was tested to 12 antimicrobials by disk diffusion agar method. We have observed that day-old chicks were rapidly colonized by new antibiotic-resistant patterns shortly after treatment with lincomycin associated with spectinomycin. After medication with the second (sulfadiazine with trimethoprim) and third (tylosin) antimicrobials, a more radical displacement was observed, and, concurrently, antimicrobial resistance phenotypes have become more complex. In contrast, more than 70% of the strains isolated in control group during the experiment displayed exactly the same resistance pattern found in the day-old chicks. This study provides clear evidence that a sequential medication of a broiler flock, with different antimicrobial classes during short periods of time for prophylactic objectives, was accompanied by a dramatic increase in both antimicrobial resistance rates and phenotype diversity of E. coli strains.

Antibiotic Resistance of Enterococcus spp. Isolated from Wastewater and Sludge of Poultry Slaughterhouses

Antibiotimicrobial resistance was investigated in 537 Enterococcus spp. isolates recovered from 22 samples of crude inflow, treated effluent and sludge collected in wastewater treatment plants of eight poultry slaughterhouses of Portugal. No significant differences (P > 0.05) were found in the resistance to each antimicrobial agent with regards to the origin of the sample (inflow, sludge and effluent). Many of the isolates displayed resistance to tetracycline (85.7%), erythromycin (45.7%), nitrofurantoin (34.0%) and rifampicin (17.8%). Resistance was also observed, but to a lesser extent, to ciprofloxacin (10.2%), ampicillin (8.0%), chloramphenicol (4.6%), vancomycin (0.9%) and gentamicin (0.4%). Resistance to three or more antimicrobial classes was present in 37.1% of the isolates. Wastewater treatment resulted in viable enterococci decrease between less than 1 log and 4 log; nevertheless, more than 4.4 × 105 colony forming units (CFU)/100 mL were present in the outflow of the plants and thus resistant enterococci are not prevented from reaching the general environment.

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin.

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P<0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.