Staphylococcus aureus Genetic Lineages Found in Urban Effluents and River Water

Methicillin resistant and susceptible Staphylococcus aureus (MRSA and MSSA respectively) remain a public health concern as human pathogens. Presence of MRSA and MSSA in river water and urban effluents was studied to analyze the S. aureus population and determine the genetic diversity and predominant genotypes obtained by spa types and MLST on each ecological niche. MRSA proportion in urban effluents was higher than in river water (P<0.05). According to the Simpson’s Index of Diversity based on spa types, MSSA isolates were more diverse than MRSA isolates (P<0.05). Predominant spa types and STs detected in MSSA river water isolates were different from those found in urban effluents. In the MRSA population, ST125-t067 was the predominant genotype detected in both urban effluents (67.6%) and river water (82.4%). Overall, the MSSA and MRSA lineages most frequently found in river water and urban effluents were human associated clones (ST125-t067, ST5-t002; ST22-t032, ST30-t012 and ST15-t084). These results show the potential role of water in the S. aureus maintenance and dissemination. Association of isolates from the river with human ones could be reflecting the effect of anthropogenic activities in the ecosystems, which highlights the need to evaluate the circulation of pathogens in the environment via water.


Introduction
Methicillin resistant and methicillin susceptible Staphylococcus aureus (MRSA and MSSA) remains a public health concern as human pathogens [1].Different genetic lineages have been described as Hospital Associated-MRSA (HA-MRSA), Community-Associated-MRSA (CA-MRSA) and Livestock Associated-MRSA (LA-MRSA).Infections caused by HA-MRSA isolates are normally related to risk factors such as hospitalization, surgery or indwelling medical devices [2].CA-MRSA affects to otherwise healthy people and infections have been linked to the presence of the toxin Panton-Valentine leukocidin or PVL [2].Finally, LA-MRSA has been considered an occupational risk although its frequency of isolation is increasing in countries with low prevalence of MRSA [3].Genetic differentiation between these groups is getting more complicated due to the incidence of HA-MRSA in the community and vice versa and due to the transmission of MRSA between humans and animals [4].Direct contact was pointed out as the most feasible transmission route of S. aureus [3].However, colonized individuals might discharge bacteria into urban effluents and recreational water [5,6].Wastewater treatment plants have been described as reservoirs for MRSA, and hypothetically, participate in their dissemination through sewage treatment plant effluents, as part of the S. aureus population might survive the wastewater treatments [6][7][8][9].Moreover, the presence of MRSA in river water [10] points out the potential role of water in the dissemination of MRSA, and in consequence, into associated environments [8,9,11].These facts led us to study the presence of MSSA and MRSA in urban effluents and river water to assess the genetic diversity and predominant genotypes within each ecological niche.

Samples origin
One sample of urban effluents was taken in July 2011 in a sewage plant that gathers wastewater from several urban collectors (untreated wastewater) in an urban nucleus with 3.2 million people (http://www.ine.es/SID/Informe.do).One river water sample was taken in September 2012 in the countryside, downstream the municipal term of a city with 8,392 people (http://www.ine.es/SID/Informe.do).

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(one per subsample) were selected as potential S. aureus and confirmed as MRSA (mecA or mecC positive) or MSSA (mecA and mecC negative) as described above.Confirmed S. aureus were characterized by spa typing sequencing the variable fragment of protein A [12], and spa types were analysed by the minimal Spanning tree algorithm (Bionumerics 6.0).Simpson's Index of Diversity (SID) and Jackknife pseudo-values (CI: 95%) were used to estimate the genetic diversity of S. aureus isolates based on spa types (Figure 2; http://darwin.phyloviz.net/ComparingPartitions/index.php?link=Tool).Multilocus Sequence Typing (MLST) was performed to at least one isolate per spa type and isolation route (n=103) to obtain the sequence types (STs) according to the protocol described before [13].Detection of Panton-Valentine leukocidin (PVL) was also carried out [12].

Statistical analysis
Fisher's exact test (SPSS 20) was calculated to analyze the relationship between the type of sample (urban effluents or river water) and the presence of MRSA and between the type of sample and the most frequent spa types and STs in the collection (n>5 isolates).

Results and Discussion
MRSA protocol detected 96 MRSA isolates out of 100 subsamples in urban effluents, meanwhile only 33/100 MRSA in river water (Table 1).All isolates obtained by this protocol were mecA-MRSA.On the Baird Parker protocol, most of S. aureus isolates were MSSA (Table 1), but some MRSA were also detected (5 isolates mecA-MRSA and 1 isolate mecC-MRSA in urban effluents and 1 mecA-MRSA in river water).The low detection rates of mecC-MRSA compared with mecA-MRSA is in agreement with other studies [13][14][15][16].However, the detection of mecC-MRSA in water effluents is of interest considering the potential for zoonotic transmission [17] and wildlife-environmental interactions of mecC-positive MRSA [10].
Only one isolate MSSA from river water was positive to PVL (ST737spa type t4801).Some studies described that PVL is increasing in the south of Europe and in some areas in Spain, but those are referred mainly to ST8 and ST80 [18,19], STs whose isolation frequency in our study (Table 1) was low (ST8) or undetected (ST80).
A higher proportion of MRSA isolates was detected in urban effluents (102/169; 60.4%) than in river water (34/115; 29.6%), differences statistically significant (P<0.05).This higher frequency of isolation of MRSA in urban effluents compared with the river water might be related to the higher concentration of antimicrobial resistant bacteria in wastewater and the population density in the area of sampling [20,21].
Isolates were grouped in 81 different spa types (Figure 1) and 42 STs, with 12 spa types and 14 STs being common to both environments (Table 1).Ten new spa types and 7 new STs were firstly described in this study

Forschen Sci
O p e n H U B f o r S c i e n t i f i c R e s e a r c h Citation: Porrero

Methicillin resistance Sequence Type [ST] spa type [number of isolates]
River water Urban effluents  Open Access

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(Table 1).Forty-two different spa types were detected in MSSA isolates from river water and 35 in urban effluents.On MRSA isolates, the number of different spa types detected from river water and urban effluent were 7 and 13, respectively (Table 1).This genetic diversity observed in the bacterial population of MSSA and MRSA isolates would reflect the S. aureus population in both water samples.
Regarding MRSA isolates from river water and urban effluents, SID values were 0.326 (95% CI: 0.102-0.550))and 0.530 (95% CI: 0.412-0.648)respectively (P>0.05).This low genetic diversity is due to the existence of predominant genotypes that included most of the MRSA isolates.In particular, the genotype ST125/spa type t067 represented the 82.4% (28/34), and the 67.6% (69/102) of the MRSA isolates from river water and urban effluents, in that order (Table 1).ST125-t067 has been geographically highlighted in Spain representing the major MRSA genotype associated with nosocomial infections [1,24].Other MRSA genotypes such as ST22-t032 and ST5-t002 (Table 1) have also been associated with human infections [4,22,24].LA-MRSA were found in river water and in urban effluents, although typical genotypes such as ST398/ spa t011 or its single locus variant ST1094 were only sporadically found in our study (Table 1).These results are likely due to the limited impact of animals in the areas of sampling, close to urban nucleus.
Our data demonstrated that the predominant MRSA and MSSA genetic lineages detected in urban effluent and river water were human associated genotypes.This is likely associated with the potential of colonized individuals to constantly release S. aureus into the environment [5,6,20], together with the capacity of S. aureus to persist in the water environments [6,10,25].

Conclusions
Our data emphasize the potential role of anthropogenic activities in the S. aureus dissemination throughout the water, and highlights the need to evaluate the circulation and persistence of this pathogen in the environment and its possible impact for public health.
n H U B f o r S c i e n t i f i c R e s e a r c h Citation: Porrero MC, Valverde A, Mateos A, Cantón R, Gortázar C, et al.Staphylococcus aureus Genetic Lineages Found in Urban Effluents River Water.Int J Water Wastewater Treat 2(2): doi http://dx.doi.org/10.16966/2381-5299.117

igure 1 :
Clustering of spa types by minimal Spanning tree algorithm.Lines/numbers between circles represent the genetic distance between different spa-types.(*): spa types detected in river water; (^): spa types detected in urban effluents and ( ): shared spa types n H U B f o r S c i e n t i f i c R e s e a r c h Citation: Porrero MC, Valverde A, Mateos A, Cantón R, Gortázar C, et al. (2016) Staphylococcus aureus Genetic Lineages Found in Urban Effluents and River Water.Int J Water Wastewater Treat 2(2): doi http://dx.doi.org/10.16966/2381-5299.117

Table 1 :
Spa types and sequence types [STs] of S. aureus found in river water and urban effluents.